Medical

Hypotheses

GENERAL

4: 1, 58-77,

THEORY

ERHAN,

Semih

1978.

ON THE CONTROL

2101 Chestnut

Street,

OF CELL CYCLE

Philadelphia,

PA. 19103,

USA.

SUMMARY

A. Cell cycle control in normal cells: The theory effects

of replication

proteins.

trigger

The former

can act either

acts only on receptors receptors

found

movement, control

within

whereby

element

the membranes;

and particularly

B. Differentiation

leotides. ferentiation

is a result

a maternal

mosomal

important

any agent - physical

against

proposes

and further

a threshold

The flawless

leads to the destruction

of these

DNA replication

- that interferes

is triggered

between

formation

histones

that:

a. Dif-

by a decrease

of

consequence

and non-histone

are not needed receptors

quantity,

cell division.

of these glyco-protein

existing

chro-

for daily activi-

in sufficient

and subsequent

with the formation

of the already

cyclic nuc-

suggests

by CAMP; d. Aging is an inevitable developing

of

d. MI acts as a fine

level; c. The initial event is the induc-

etc.) of the genes that

unscheduled

them;

and they

inhibition

e. Both RT and Ml act through

of the model

is stimulated

a. These membrane

is due to contact

RT from binding

of tissues;

interactions

that:

glyco-

while the latter

and can bind each other

of proliferation

- below

receptors

symmetry

and prevent

-S-S- bonds

ionic-,

or chemical

as well as that

is a potential

and this event

of the antagonistic

The model

in all cells; b. Differentiation

mRNA

C. Neoplustic changes:

critically

lecules

- a particular

(crosslinking,

ties of the cells. becomes

found

and is due to secondary

proteins

control

regeneration

of the asymmetry

methylases

of differentiation

membranes.

bind each other

on the premise

or on the membrane

have molecular

c. Primary

during

is based

( MI ), both of which are, in all likelihood,

and aging: This is an extension

macro-molecule

of histone

the cellular

b. RT receptors

RT receptors

developed

inhibitor

on the chromosomes

inside

are glycoproteins;

can move freely

tion

( RT ) and mitotic

membranes

Hence

receptor

or membrane

mo-

receptors

carcinogen.

CELL CYCLE

CONTROL

IN NORMAL

CELLS

INTRODUCTION

The finely

tuned

and extremely

well coupled

“ life ” , appears

to be directed

for self perpetuation.

sent urgency scheduled bryo.

of living cells to devide,

cell division.

The potential

is tempered

This we observe

for division,

series of reactions

at the end of the differentiation.

A cell’s life starts

leading

of the genetic

round

of cell division

is usually process

much

shorter

than

place. There

the previous

giving rise to two daughter

with cell division,

material

by a concern

is, again, a seemingly one. Cell division,

cells. It is obvious

over the control

followed

quiescent

which

period

em-

that emerges

by an apparently has to occur

quiescent

before

the next

even though

this one

is also called mitosis,

that this phenomenon

of un-

in the developing

in the higher organism

of the cell, which

called

itself by the ever pre-

of differentiation

is not lost but only masked

period

can take

which expresses

in higher organisms

as early as the appearance

however,

to the replication

which gives rise to the phenomenon

This tendency

is cyclical,

terminates

this

hence the concept

of cell cycle.

The cell cycle is customarily divided into four unequal parts, all of which are experimentally ( S ) could be seen and identideterminable: Gl , S , G2 , D . Both cell division ( D ) and DNA replication 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

precursors duced

necessary

during

for the replication, G1, which can be looked

activities

culminate

stimulate

DNA replication

with

the formation (1,2,3)

of an explanation.

Now we know

that the

be they low molecular weight intermediates or enzymes, are proupon as growth period. G2 is also a very busy period, where the of the mitotic

or inhibit

apparatus.

cell division

(4,5).

Many

factors

Chemically

most

have been of them

isolated appear

that to be

glycoproteins. Thus

the events

that

control

the cell cycle

assume

a dominant

58

role for the well being of the organism.

This section

will describe

deal with the problems

a model

for the control

of differentiation

of cell cycle of higher

organisms.

The next section

and aging and the last part will try to analyze

will

what causes neoplas-

tic changes.

The model 1. All cells are capable molecules shold

of producing

are glycoproteins.

prenomena,

level, enabling

a replication

RT is universal

i.e., they

can only be effective

them to tie up a certain

fraction

2. TR can function

in two

found

of cell membranes.

on the outside

trigger

different

(RT) and a mitotic

while MI is tissue specific.

ways:

when

their

inhibitor

concentrations

of the membrane

(MI); both of these

Both RT and Ml effects exceed

are thre-

a minimum

threshold

membrane

receptors

receptors.

a. on the cell chromatin,

b. on specific

3. Once synthesized inside the cell RT binds to replication initiation region of the chromosome(s). the end of DNA synthesis replication machinery dissociates and RT leaks out of the cell and becomes “ humoral factor ” which can act by binding to the membrane receptors. 4. In higher thelial,

organisms

mucosa

circulates

RT is produced

cells etc.,

in the blood

due to normal

stream

are found

cells can bind each other 6. Genes

coding

because

They

it can act directly

on chromatin

7. In higher

the cell cycle

organisms

by tying up the membrane

8. Superimposed upon

cellular

factor

such as bone

above

needed

provide

marrow,

intestinal

the humoral

to replace

the total

bridges.

toward

inaccessible

epi-

factor

cells, in various

that

tissues,

RT effect

are free to move

so that receptors

on adjacent

of differentiation

while MI is produced

simultaneously

continously

in a cell, RT prevails

its own specific

receptors

over Ml found

in-

lasts only for one cell division.

is primarily

receptors

circumstances

to RT.

When produced

controlled

by contact

limits the accessibility

exerted

the end of wound

concentration

MI synthesis

certain

complementarity

while MI has to act through

on this is the control

effective

and under

dimensional

off at the beginning

life of the organism. However,

dent

tissues

as described

the cell membranes

and make the receptors

for TR are turned

the entire

particularly

by certain

released

have three

side the cell membrane.

which,

continously being

and acts as the initiation

outside

the membranes.

throughout

after

turnover.

5. RT receptors throughout

which

At the

of MI produced

as well as methylation

by MI which healing

inhibition

of these receptors

acts as a fine control

and tissue

regeneration.

by each cell and collected of histones

of movement

are triggered

( CI ),

to RT. of cell division

This inhibition in the tissue,

and is

is depen-

through

inter-

by CAMP.

Discussion The cell cycle of all cells appears 1. An RT probably

to be controlled

acting at Gl/S juncture

2. An MI acting at G2/D juncture All living cells are capable

at two points

by two different

factors:

of the cell cycle (1,2,3,6,7,8)

(4,5,9,10).

of producing

both

RT and MI at a constant

59

level. RT is a glycoprotein

synthesi-

zed under brane

the control

and binds

tion related

of a specific

to the

proteins

initiation

and becomes

the complex

the humoral

factor.

the cell membranes. etc., and provides

may share

a short

amino

acid sequence

the membrane

have

to stimulate

reported

of DNA in those

cells which

in various

I propose

tors,

tissues.

which

are found

synthesized into

mitogen

receptors,

the receptors cosylceptor

together

MI stimulates

these

why so many seemingly

in various

systems.

in order

to replace

receptors

to which

This factor

rate of synthesis

their

synthesis

(11).

receptors

found

binding

(19,20). cyclase

(G-cyc)

are found

turnover

The receptors

for RT are

the end of GI (16). They are

inside

is also very fluid and (17,18).

together

and CAMP phosphodiesterase

proteins replication

as well as the MI recep-

the membrane

the cell membrane

enzymes

to initiate

The cell membrane

to move within

site outside

Two other

toward

of binding

unrelated

due to normal

(12,13,14)

are also glycoproteins.

is completed.

have been shown

helps

the cells dying

RT binds

(15)

after

for MIS, which two

are tissue specific

enzymes:

I propose

that

with two enzymes:

the membrane

(A-PDE).

within

All of these

glythe re-

enzymes

are

adenyl

cyclase

glycoproteins, (A-cyc)

have their binding

site inside the membrane,

and cGMP phosphodiesterase

(G-PDE).

Binding

of

these enzymes.

to G-cyc

the influence

increase

This may explain

with the maximum

for RT have their

with

Proximal

it can bind to the cellular

the cell membrane

for instance,

guanyl

conformation

when RT binds to the receptors.

The receptors

When

the membrane

complex:

mem-

and the cell membrane

as well as a few sugars with RT, may also be capable

have to divide

inside

and sialyl-transferases

stimulated

three dimentional

the RT leaks out of the nucleus circumstances

receptors. replication

that

in the GI period,

inserted

in a unique

the nuclear

The rest of the replica-

In higher organisms RT is produced continously by some tissues, such as bone a steady supply of humoral factor. Some glycoproteins other than RT, which

and thus stimulating been

these

it penetrates

on the chromosome(s).

complex

dissociates,

Under

in the cytoplasm

of replication

then bind to this RT-DNA

At the end of replication outside marrow

gene. Once synthesized region(s)

is also a Ca++ pump

there

of RT the pump promotes

RT is bound

to a cell receptor,

of cGMP and a decrease

which

Ca++ uptake

normally

extrudes

Ca++ from

into the cell (21) (Figure

G-cyc as well as A-PDE is stimulated.

of CAMP concentration.

Ca++ taken

the cell, but under

1). This leads to an intracellular

up, which

follows

cGMP increase

by 10 minutes in lymphocytes (22), reinforces the RT effect by stimulating A-PDE and inhibiting cycl; cGMP formed also stimulates A-PDE (23). Thus the humoral factor creates conditions conducive cell proliferation:

increased

cGMP and decreased

CAMP, which

stimulates

histone

methylases

Ato

as well as

MI production. This is followed by stimulation of RNA polymerase I, then *RNA polymerase by phosphorylation of certain acidic chromosomal proteins (24) and transport protein

into the nucleus

tial for the induction wall of incipient

(25) and binding

of furrow

and advancing

to cellular

formation furrow

DNA (21, 26). Ca++, furthermore,

as it was shown

(27).

II and III (21) as well as of a particular cytoplasmic

I further

to be needed

propose

appears

for deposition

that the glycoprotein

to be essen-

of filament

molecules

in the

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 (Cl). Reother. This situation is called “ contact inhibition of movement ” or simply contact inhibition ceptors engaged in CI are no longer available for humoral factor. All of the membrane receptors cannot

and need not to be engaged the available

receptors

upon the source

in Cl, as the effect

that need to be engaged

of RT is a threshold in CI, to prevent

phenomenon.

untimely

stimulation

The actual

fraction

of

by RT, may depend

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

60

c;a - +------

5’-AtiP /

Fig. 1.

Portion inhibitor;

of cell membrane RT: replication

phosphodiesterase; pump. b+>

showing

elements

FIGURE

LEGEND

which

participate

in the control

of cell cycle.

trigger;

G-PDE:

cGMP phosphodiesterase;

Gcyc:

Acyc: adenyl : inhibition;

cyclase;

GT: glycosyl

ST: sialyl transferase;

F>

: stimulation.

61

transferase;

guanylcyclase;

MI: mitotic

A-PDE:

CAMP

Cap: calcium

MI can

be monitored.

The

(28), the MI molecules When CI occurs

optimal

tissue

concentration

which are lost being promptly

the binding

sites of the receptors

a drop in cGMP concentration

of MI is maintained

become

through

cell-cell

bridges

by de nozw synthesis.

replaced

inaccessible

to RT, which

immediately

leads to

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

cell division

and together

of RT is good

The presence

of tubulin

cytochalasin

in various

B, colcemid,

a single cell division

to cause paracrystalline

mouse

lymphocytes

chicine

binding

(30,31).

has been. demonstrated

(agents

aggregates

(corresponding

activity

tissues

vinblastin

shown

CHO-Kl

for only

Unless RT is present

continously,

stops after one cycle.

known

to disrupt

of tubulin.

Supernatant

to a membrane

(34). The presence

cells have also been proposed,

enriched

(32,33). fractions

of these

on the reversible

were

changes

elicited

within

was

of purified

also shown

systems

cells with

systems)

of the homogenates

preparation)

of microfilament-microtubule

based

Treatment

microtubule/microfilament

to have col-

the membranes

by colcemid

of

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 epithelial-like appearing cell membranes, leading of colcemid to these cells, then, converted (35.) Polymerizing During

effect

proliferation

bule systems

finally to fibroblast-like appearing cells showing CI. Addition them to epithelial-like cells with throbbing knobbed structures

of CAMP on microtubules,

the cells are most

of the membranes

however,

has not yet been demonstrated

likely to be in an epithelial-like

are not organized.

During

conclusively.

form, where microfilament-microtu-

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 concentration

increases.

I propose

of CAMP. During

of cells to be aggregated The receptors tion,

microfilament-

that have not been engaged from

replication.

the cell surfaces I also suggest

nes which increases

Application

microtubule

a maximization

systems

of the cell-cell

are assembled

contacts

under

is achieved

the influence

and the tendency

by Con A is reduced.

were it not for a nondialyzable

be removed duled

that

this organization

the affinity

in CI could

heat-, by dilute

trypsin-

urea treatment

that an increase of histone

get involved

in Con A or PHA-mediated

and cycloheximide(36)

which

in CAMP concentration

binding

sensitive

molecule

decreased initiates

agglutina-

which

the chances the methylation

can also

of unscheof histo-

to DNA.

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 supplements 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 efficiency. 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 enriched

medium,

such

cells may undergo When confluence which is present

as 3T3 cells,

a limited

may be lacking

cell division

in the protein

removable

by dilute

urea treatment

and

for one cycle.

is distrubed, say, by a policeman, in the medium, initiates replication

the number of exposed receptors and cell division. This continues

62

increases and the RT, until new cells produ-

cd

come into contact

with each other.

2. IUI ~KSUP\: When some

cells die, due to normal

degraded

cells

leaving

and a limited Wound

certain

cell division

healing

with

exposed

will follow

represents

a similar

that

tissue cells reach each other,

may

touch 3.

not be exposed

another

tissue,

The major

Kr~~c~ten~ticin:

cell division difference

from

when

normal

stops.

dividing,

( a detailed

discussion

difference

between

wound

is destroyed

receptors

will also be

to humoral

by the wound

which stimulates

occurs

cell division.

at the surface

of a tissue

will be given below

healing

factor

and then

When new11

cells and the third

does not

regeneration).

is the need to stop

This, of course,

section

any cells

which

for tissue

and tissue regeneration

size of the organ is reached.

cells vs. the neoplastic

their

be accessible

Here MI may also play a role by blocking

that

preoperative,

contact

factor,

cells, a situation

the original,

between

cell-cell

to humoral

cell division

to other

will then

of dead cells is completed.

First

situation.

and are autolyzed,

which

until replacement

the cells at the site of loss of Cl are exposed formed

turnover,

receptors,

is also the major

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 released by intracellular Of course

MI receptors

the cells that

and MI concentration

are found

tion,

and,

a. Cell-cell

b. Cells become

c. During

the

tocytes,

and hepatocytes cells are inhibited

Ligation

of a portion

is eliminated

division

triggers

MI available

normal

factor,

cell division

by Cl,

the following

events

releasing

leading

DNA synthesized

to exponential (38).

of cells increases,

c. When total

the inhibi-

occurs

80% of this replication

cell division,

of humoral

from

replication,

75% of total

for about

cell division

prevented

the cells at the regions

which

about

slows down

liver reaches

from

receptors.

of liver is removed

by CI, d. As the number

hepatectomy,

the influence

When the remaining

factor

1 and 2 account

of liver limits the total under

are still primarily

hepatectomy,

and gradually

by liver before

tion to cell division stream

in zones

to other specific

cells. When a portion

to humoral

after

from further

increases

the level produced

a tissue

contact

accessible

72 hours

formed

of MI procluced

inside

by their binding

acts on the peripheral

are likely to take place: growth,

are measured

in hepa-

The newly

the total amount

MI concentration

reaches

stops. to the remaining which readily

tissue thus releasing reaches

size, total Ml concentration

there through

is restored

the inhibithe blood-

and further

growth

is prevented. .4fter single-nephrectomy

a similar

situation

prevails.

The body

a certain

chalone.

When one kidney

is removed

level of kidney

until regeneration For all these derepressed;

taking place in the remaining

processes they

take

to take place

place,

because

genes were activated, and there a situation known as neoplasia.

there

kidney

restores

of the organism the inhibition the original

is tuned

to function

of cell devision

Ml concentration.

is no need for the RI genes of the differentiated

of the cascade

are circumstances

of events

under

63

which

that

leads

this takes

with

is released,

to gene activation. place,

then

cells to bc If the R-1‘

one is faced

with

DIFFERENTIATION

AND AGING

Introduction As mentioned that

above,

the tendency

is seen so forcefully

Evolution

had selected

organisms

controls

ted

cells

through

Since most tween

path

whereby

cell division

by repression

the

specific

proteins

to describe

found

which

microorganisms,

is the overwhelming

has been

this tendency prevail

of genes,

interaction

in all but a few tissues.

by nuclear

and unrepressed

of all living cells higher

organisms.

constantly.

Hence

in higher

This suppression

occurs

primarily

nuclear

proteins,

genes.

starting

force

for day to day activities

basic and acidic

the events during differentiation,

driving

in differentiated

to divide is checked

is protected

on repressed

tempered

which are not needed

between

of the DNA of higher organisms

nuclear

attempts

the

against

by CI and secondarily

to divide,

among

proteins

of differentia-

and cellular

DNA.

there must be a difference

This section

will present

with this difference

be-

a model which

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 temporal

order

produced

by the first cleavage

of the synthesis

3. The decrease

in the concentration

the initial event in differentiation: 4. Differentiated (Figure

of a maternal

the formation

cells have negative

is expressed

membrane

control

by the components

components

controls

macromolecule

of histone

of the cell surface.

the progress

to a critical

The

of differentiation;

threshold

level triggers

methylases;

on cGMP synthesis

and positive

control

on CAMP synthesis

2) ;

5. CAMP stimulates 6. Phosphorylation related

of various

alkylation of histones

of histones; is involved

in cellular

events

leading

to cell division

while acetylations

are

to transcription;

7. Fine tuning

of transcription

8. MI gene is turned

is controlled

by methylation

of DNA;

on.

Discussion Any discussion of differentiation becomes very quickly unmanageable ground rules. This is so because the methods used to analyze differentiation the interpretation of the results obtained. For instance, there is a dogma

unless one agrees upon certain can to a great extent influence 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 genes and that the differentiation is the expression of new genes which were repressed earlier ferentiation of systems which differentiate as separate elements, such as blood cells or pigment follow different rules with respect to division and differentiation than do tissue systems. If on

64

available (48). Difcells, may the other

(a)

FIGURE

Fi,y 2,

a) Control diesterase. b) Control adenyl

of cGMP concentration during Activity in arbitrary units. of CAMP concentration

cyclase.

Activity

in arbitrary

LEGEND

cell cycle. Solid line:guanyl

during

cell cycle.

units.

65

Solid

cyclase;

1ine:cAMP

broken

1ine:cGMP

phosphodiesterase;

phospho-

broken

line

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

cific to differentiated it was found ses rapidly matin

that

(48) - very early during

” by some

cells, the idea of “ quanta1

during

( blastula

) supports

that differentiation the later stages haemoglobin,

Based on the observation

transcription

is a process

2,2 times

of decreasing

of differentiation

that were believed

to be spe-

” loses its meaning (505152). In one study of X. laevis collagen synthesis starts at gastrulation and increa-

the development

up to the larval stage (53).

differentiation

division

better

template

that in Drosephila

embryos,

than later stage chromatin,

activity

may lead to the synthesis

and hence increasing

of specialty

earlier

chro-

one has to conclude repression

molecules,

even though

such as actin,

myosin,

etc. (54).

Thus I favor the following 1. Differentiation proliferation

view in my model:

follows

proliferation

of the differentiated

2. Even though

the synthesis

embryogenesis,

during

However,

embryogenesis.

cells. These can still undergo of some

special

molecules

more and more genes are turned

this does

not

may

not

be observable

off as differentiation

during

the internal

structure

earlier

stages of

progresses.

Coming back to the discussion of the model, one may ask: “ is there any justification larity in the egg cells?‘. My answer to this question is an unqualified “yes”, for a myriad To begin with one may consider

rule out further

differentiation.

of a cell. The contents

for the idea of poof reasons.

of a cell are not like a dilute

solution but a semisolid gel (55) where endoplasmic reticulum, mitochondria, ribosomes etc., are distributed unevenly throughout the cytoplasm. The only way one can conceive of a cell as a symmetrical body is if the contents trical fashion. object.

of a cell were distributed

It is impossible

Thus division

In invertebrates,

the cytoplasm egg within

expresses

itself

is known

as “ localization

in the uneven

(56).

Isolated

from

adjacent

least

fist (58) in the form

plane

blastomeres

can be expected during

distribution

were shown

throughout

asymmetry

embryogenesis,

in developing

to develop

by the metaphase

to continue

where

expresses

especially

during

signal carriers

- channels

into their

certain

particular

as cleavage

occurs.

the complete

symme-

symmetrical

cells. of the cytoplasm egg already

region(s).

This phenomenon

which

cell linage of various organisms

final shape and size, independent in such vertebrates

What

orderly

gastrulation,

apparatus,

a polarity,

I am suggesting

shifts

poses-

This polarity

of the input as birds and te-

Since cleavage planes are always established

embryogenesis. That

ellipsoidally

Even the unfertilized

plate of the mitotic

itself.

through

hence asymmetric

has also been observed

teins has been proposed (59). It is conceivable each other, such as the ones found in Volvox, as primitive

through

of yolk and active cytoplasm.

occupied

(56).

of the cytoplasm

” has been exploited

speaking

as an ellipsoidally

polar and one sees a localization

fertilization

is able to enter

cells (57). This kind of polarity

previously

as the stage

sperm

after

an ovum,

yield two unequal

of egg cells is definitely

a few minutes

in one axis so that

or more generally

any real cell, including

of such a cell will invariably

of an uncleaved ses polarity

in a spherically

to consider

of individual

may be due to differentials

in the

once established,

is to consider

the ovum

cells and cell complexes of the cell surface

pro-

that during evolutions, synchrony of cells found close to preceeded differentiation; cell contact points functioning

which RT might be transported

- between

cells.

Recently the clustering of Con A receptor sites on certain cell types in early embryos has been demonstrated (60). The mobility of specific migratory cells was found to be very similar to the mobility of the invasive malignant Secondly,

a glance

cells (61). at a series

of photomicrographs

showing

66

the progress

of cell division

in a mouse

egg

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 divides,

the one that

touch

only three

that

was in contact

one cell, because

were to have certain could

constrain

by internal much

of preceeding sites which

cell factors

closer

induction

newly

while

centers

primordial

principles

may have introduced

universally

differences

evolved

only

through

to chance

ones.

Thus,

region

certain

by a parasite

accepted among

more

a particular

I propose

I am suggesting

that cell surface, should

Locke

discovered

tration

gradient that

gradient

positional

by starvation

egg, a point

may be reached

the optimum

and it may trigger propose

that the mRNA

mRNA

of positional

substance

(74). It is quite where

differentiation.

as mRNAs

(nl)

of unused

that

than to the the egg

do invade their hosts

more valid because, event,

as the previous

control

if an attack

such as fertilization,

be left

controlling

section

factor

in dif-

also had concluded

that

of cell cycle.

information

(72). A recent

from

which

triggers

the

is definetely

codes

which

determines

both

that this gradient

computer

simulation

based

of certain

that during

cell division,

of certain

This situation hand,

a critical

polarity

behaved

and the

like a concen-

of this gradient

transcription

histone

on the premise

following

from

the concentration

phenomenon

into a flagellated

form

the fertilization

of an

the outside

of many

found

found

of various

genes

maternal

that histones and progression

to cells

macromolecu-

I

of differentiation.

) is the key molecule

a preset

and particularly

the binding

” of those

during earlier stages - morula

during gastrulation

in each cell falls below

is not sufficient

“ starvation

and thus act as a trigger

( fI histone

by increasing

for the initiation

nutrients

protozoa

may be considered

threshold

( fI histone

its concentration

Lie nova

Differentiation

likely

for fI(m)

the fI (g) histone

and hence,

(73).

On the other

(75) may fall below

due to cell division,

genes

later

can only penetrate

of egg cells, is the major

It was later shown

the diffusion

Histone methyiases, initiate differentiation toward the DNA of various genes. This model

inclusive

cell (71).

rate of cell division.

les such

is different

evolution principles,

in their expressions,

make this argument

how can an important

appli-

the first

basic biochemical

these

The fact that most bacteriophages

for the precise

information

is preceded

which

organisms,

that

from

“ set ” at some stage in the cell cycle to the ambient concentration to a concentration gradient of a diffusible substance these set values are necessary

in addition

and when,

be a model

stemming

that certain

likely

becomes fields, with

ceils may be come

to have complete

support

important

of a diffusible the

gradient

laws, even though

it is more

not be too surprising

fate of the epidermal

suggests and that

an axial

(63). If one also

at any point on the egg membrane?

This conclusion

developmental

one has to conceed

all eggs are polar and sperm

controlled,

In summary,

were critically

that two interacting

to the lower

areas on the cell wall (69,70)

ferentiation. the membranes

stages

studies be caused

pole, the simulation

the idea of all organisms

Furthermore

that

simulation

can entirely

to the main axis of the egg, might

(67,68),

them.

If egg membrane

Computer

the earlier

to all living cells as inviolable

of the egg membrane.

of the sperm

during

still

suggest

then the cleavage planes

pattern

near the animal

Finally

when

will be closer

has to be so precisely

penetration

Today

will have to apply

to all living organisms,

highly

(64).

property?

each other.

are not involved

sides with respect (65,66).

are common through

cells will touch

results

or does it perhaps

for instance,

the view that differentiation

obtained

in general

cell is nearly

the poles,

field with the maximum

on two opposite

cable to morphogenesis

contacts

to have this unique

to become

divided

with five cells while five others

of an accidental

was destined

cell interactions

of a gradient

to the experimentally

and biological

events,

of the L,yr~neu egg supports

the presence

now has contacts

observation

were destined

the areas whereby

of embryogenesis assumes

with four others

cells. Is this a coincidental

affinity,

are the molecules of differentiation.

here

level, this need for fI of histone

of those

responsible

methylases.

histones

affected

for repression

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 these

as well as interactive

modifications,

differentiation.

histones

Goodwin, be necessary

of accessible

cell states

of cell types

for arriving

well correspond

states.

synthesis

occurs

found

by the methylation

toward words,

concentration

is controlled hand,

of mRNA,

seen during

which

low f2b/

stage, has already

f2a ratio of histone

the heterochromatization converted

to fl(g)

codes

for fl(m) begins

of a different

on, when

histone

and that

their

amino

I propose, affinity

needed.

of

that in differentia-

negative

control

Adenyl

( Figure

2).

cGMP

cyclase ( A-cyc )

The intracellular

(79).

while

activity

falls below

together

fl(m)

This observation

in the template

The difference,

CAMP continously.

a critical

level is

threshold

with the transcription

up to morula RNA isolated

stage, than the fl(g) from morula

level,

of histone polysomes

directs

from gastrula

lends support

to the view that f2a is involved

as the embryo

of the chromatin.

acid sequences

were

polysomes

me-

which is

RNA isolated

of the genes as well as the view that

trula there will be a decrease

is under

of hisbetween

( A-PDE ) on or off, as needed.

of histones,

synthesis.

may very

at a low level and the intracellular

( G-cyc)

been established

of a high f2b/f2a/ratio

required

proteins

and thus the binding

by CAMP. I also propose

) is active

stage, de nova RNA synthesis

gastrula

( eu- ) chromatin.

cells, and furnishes

CAMP phosphodiesterase

with the

processes

(77) and since the synthesis

the basicity

is stimulated

cyclase

in the number

(78) there must be a difference

and CAMP production

guanyl

on in these

decision

and nonhistone

DNA synthesis

increase

( G-PDE

for the first time. The synthesis

the synthesis

which

a reduction

during

specificity

(76).

) and unrepressed

positive

by turning

the quantity

at the end of morula thylases,

( hetero-

needed

to a value commensurate

histone

stages of embryogenesis

of histones

is under

is always

by switching

when

with

even without

control

that low repressor

of the embryonic

among

by Goodwin

cGMP phosphodiesterase

on the other

Thus

interactions

DNA, and that this methylation

ted cells cGMP production

controlled

and a stabilization

described

in the repressed

conclusively

of possibilities

Actually

tuned

goals simultaneously:

number

contemporaneously

is furnished

In other

organism

for this purpose.

a very finely

demonstrates

two complementary

the earliest

the histones

be necessary

of furnishing

analysis,

The well known

takes place even during

histones

may

the astronomical

to the interactions

Since histone tones

to achieve from

in a higher

at these

that

be capable

in an elegant

may actually number

terms

may

moves

from

It was also found

different

and most

directs

morula

that fI(m)

importantly,

a in

to gaswas not 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 (54).

embryos chromatin. cation.

Blastula

There

chromatin

is sufficient

The phosphorylation

preceding by histone

S phase

was also shown evidence

of fl was found

(8081).

Of special

V (82). Since the nuclei

synthesis,

histone

V would

eliminate

V seems any

to be the most

throughout

the cell cycle

(84).

synchronized

cultures

phorylation

may

Phosphorylation Acetylation synthesis. tomy)

that very

of histones,

In calf thymus

were

occured

on the other

hand,

maximum

cells, incubated

with

activities.

was found

of repli-

fl is replaced DNA and RNA

through

by histone

phosphorylation

of

(83) while it was not methylated to any appreciable

fraction

of f3 (80)

degree

and only in

At this stage of the cell cycle, f3 histone for realignment,

phosetc.

of RNA synthesis.

histone

in cells actively

acetylation

( 6 hours after operation)

only arginine-rich

68

than gastrula

as removal

to occur primarily

arginine-rich

RNA synthesis

minor

mitosis.

in both

This way replacement

processes

hand was not phosphorylated

to be independent

l4 C- acetate,

inactive,

of all histones

such

activity

with the initiation

that in avian erythrocytes

in a slow moving

function,

rat liver the maximum

template

GI phase of cell cycle but immediately

of these

rich in cells undergoind

was also found

to precede

both

phosphorylated

well serve a different

of histones

In regenerating

was found

which

during

are apparently

activation

f3 on the other

phosphorylation

higher

fl is involved

is the observation

by suppressing

fl was shown

histone

erythrocytes

of accidental

fI. Histone

and it was found

that

not to occur

interest

of these

to function

chance

to have 2,2 times

to suggest

fractions

engaged

in RNA

(3-4 hours post-hepatec(85). f2al

and f3 were found

to

However, labelled (86). fI and f2b were not labelled and did not contain n-acetyllysine. acetylated at their terminal amino position. Rat liver nuclei, too, they as well as f2a2, were extensively incorporate labelled acetate from acetyl-CoA into histones and nonhistone acidic nuclear proteins (87).

be appreciably

The order

of acetylation

label. Pigeon rylation

was f3) f2aI>

liver acetylase

(88). In cell culture

Thus two different

2. Acetylation various cellular Of course

histones,

fI was not acetylated

phenomena

1. Phosphorylation division.

f2b) fI. Histones

also acetylates

related

of histones

f3 (70%) and fZal (25%) comprised

where

become

if the gene activation

mode

all the

phospho-

apparent:

is going to lead to DNA replication

of histones occur if the genes are activated for transcription activities but not involved in the replication of DNA.

this is the predominant

95%of

with the highest

(89).

to gene activation

occur

f3 is the one species

of operation,

since there

and translation

are many

and cell

needed

for

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

is rich in adenine

supporting

also found

previously

and thymine

cells of Drosophiliu

Thus fI of blastula mals,

1 proposed

to the model

chromosome(s)

the

view

embryo

of its involvement

to be independent

(11)

I suggest

and the region(s) was found

that

the replication

is covered

to be quantitatively

in replication

initiation.

initiation

site(s)

on

rich histone

fl.

in the adult

ani-

of histones

was

by very lysine less than

Phosphorylation

of RNA synthesis.

I also propose that histones f3 and f2aI are involved with the repression of genes in differentiated organisms. Both of these histones appear to be bound to DNA through divalent cations (92). And fully differentiated old leaves and pith tissues were found to contain more FII and FIII histones (93); however, the than calf thymus and pea histone FIII, while the lysine/arginine ratio of FIII was found to be different same ratio for FII was similar to that of pea histone. f2aI

is believed

was found decrease

to be involved

to be increased

in the amount

in vivo to rats increases

Estradiol

given

This was found

some

was shown that

transition

arginine

residues

in histones,

methylation,

which

normally

of chromatin, morula

capacity

of uterine

in the amount

to increase

the binding

of acidic protein prior to transcription. Histone RNA synthesis when added to DNA (96,97).

There

is also another

which

has been

possibility

demonstrated

for modifying to occur,

DNA-histone

even in the isolated

f

within

3

is a

(94). 15 minutes

after

which methy-

of f3 histone.

This decrease

protein

of histone,

(95). This means

was reduced

Certain

together

to be the most

the methylation

(98,99).

that there

1 activity,

was also shown

interactions: nuclei

progresses

methylase

sulphydryl-containing

Its synthesis

suggests

chromatin

of histone

and a 58% decrease of an acidic,

in heterochromatin.

(79), which

as embryogenesis

by a 50%decrease

is expected

found

to gastrula

RNA synthesis

template

to be paralleled

to be due to de uovo synthesis

with the synthesis ficient in repressing

from

of DNA able to support

injection. lates

in the superstructure

during

cf-

of DNA bases,

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 thymine (99). However, this reaction was found not to occur in isolated under

the control

of some

cytoplasmic

regulator.

Methylation

69

occured

of these Smethyl nuclei, suggesting

preferentially

cytosines to that it was

in cytosine

isostichs

and CpG dinucleotides (100). which dissociates arginine-rich sis on the control

of mammalian

this modification tosine

duce the affinity

of histones

there

which

may

(102). Since S-methyl

the base sequence

of the DNA will be restored.

and thymine-rich

and fl

to them.

binds

be the control

already

between

mentioned

iie nova

synthesis

to contain

are many

possibilities

there which

can be utilized

regions

There

on chromosomes

are also cytosine

to guanine, normal

can be expected

cy-

to re-

of f3 affinity acidic

at the blastula

proteins

(95).

tuned

regions

bind to them. chromatin

DNA was found

Drosophila

embryos,

control

of the repression derepression

proteins.

to correlate also, were

later in development

as well as during

on chromo-

These are likely to occur

by nonhistone

rat uterine

stage, which disappears

differentiation

genes.

and f3 histones

toward

for a very finely

during

which are the sites for initiation

and guanine-rich

of individual

can be methylated

of sylphydryl-containing protein

Thus

is still complementary

of this methylation

and DNA can also be modified

the decrease

a nonhistone

genes

or wound

histones

above,

The effect

sites for the transcription

near the 5’ end of the genes, their cytosines The interaction

cytosine

of the genes and at the next cell division

f3 and fZal to DNA (98).

are adenine

of DNA replication somes

genome

does not change

concentration

In summary,

Methylation was found to be increased after trypsin treatment of chromatin, histones (101). These observations appear to support Georgiev’s hypothe-

As with

found

(54).

and derepression following

of

hepatectomy

healing.

Aging What

happens

to those

of the cells in which cent

histone

molecules

for transcription phobic

genes,

(103,104)

first

observed

being studied

correlates (105,106).

(.54,94)

Indeed,

Ebert,

had

protozoa,

of those induced

studies effect

and the renewed

the act of chromosome

replication

take place between

genes which disulfide

to undergo

bonds

and hydro-

cell division,

or even transcription work on these bonds

observed

in the onset

of conjugation formation

can be attributed clean

after

can occur, have to be been

of the reaction

of new structures

may actually

adja-

are not needed

on aging, a delay has repeatedly

to the delay

the rejuvenating

and micro-nuclei

that

during

linearly

for the day to day activities

interactions

can be ionic bonds,

When such a cell is then

takes time.

In many

of the old macroproposed

proteins

These interactions

the age of an animal

elimination too,

are not needed

that secondary

injection, etc., then, before any replication have to be dealt with. The enzymes which

and this of course

which

which

suggest

and nonhistone

as well as crosslinks.

hepatectomy or isoproterenol these secondary interactions

cells,

I would

as well as replication.

interactions

induced

in differentiated

they are found?

to the

(107,108).

inactive

genome

regions (109). Thus according to this model aging appears to be inevitable because it is the result of differentiation, since in the higher organisms control of unscheduled cell division is of utmost importance. However,

once an understanding

down the process

is reached

of the controls

of these phenomena,

one might be able to slow

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

to the proponents of this latter group, there is hope in the site of the disease. Hence, according the events leading to neoplastic state in one model and one can be reasonably confi-

being able to explain dent that this model

may be applicable

describe

conditions

how certain

to all forms

of the disease.

can alter this control

scheme

In this part of the manuscript

and lead to the changes

I shall

of hyperplasia

and

neoplasia. 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

In this way the organism

of cell membrane

insures

itself against

receptors

in necessary

unscheduled

replication

quantity

It shoulJ then ~0110~ that any event or agent that leads to an interfbrence tion ofgl_ycoprotein There

are several

or physical receptors

receptors, possibilities

agent

which

through

concentration brane

in the

RT receptors,

can be conceived membranes

destructive

of glycoprotein

receptors,

receptors

than

can affect

its physical

with the synthesis tion of surface

particularly

effect

precursors,

c. A defect

which

with

stimulation

of cell division which

defect

leading results

a. A chemical

and to eventual

b. A metabolic which

or destruc-

agent!

may lead to this interference:

of A-cyc,

e. Any condition

thefbmation

a carconigcnic

sialyl- or glycosyl-transferases

importance.

cell division.

will lead to inactivatron

on membranes,

in the formation

cells, d. A continued

or on the chromatin,

is potentially

directly

iacquire utmost

and consequent

which

to incomplete

in an unusually

by agents

that either

may lead to a shortening

loss of

can interfere forma-

low CAMP act on mem-

of Cl phase of the

cell cycle.

The model 1. Cell membrane

receptors

2. CI is thus effectively another

are affected

interfered

adversely

with,

by anyone

a consequence

of the above causes a,b, or e.

of which

is seen as a piling of cells one on top of

in cell culture.

3. Humoral

factor

in the

section

first

or quantity,

can initiate

the effect

of RT is not limited

4. CAMP concentration 5. Histone

however,

and cell division since

in accordance

RT receptors

with the model

are not present

to only one cell cycle but continues

in sufficient

described quality

cycle after cycle.

inside the cell falls to very low levels.

methylase

6. MI synthesis

DNA replication

of this manuscript,

induction

will no longer be possible.

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

71

the control

of cell cycle by their

interference

with histone

methylation

tion of A-cyc or due to a defect 10. Viral infection by producing

and perhaps

RT needed

is also stimulated. and hence

the dreepression

even certain

bacterial

of cellular

inside

of CL So chronic

activation

and nematodal

Because

RTs are synthesized

the control

These conditions

or to an irreversible

for its own replication.

Since these

are beyond

and with MI formation.

in its synthesis

infections

of the universal the cell, they

infection

may be due to an inhibi-

of A-PDE. can also affect

nature

cell cycle

of RT, host replication

act directly

on cellular

may lead to irreversible

chromatin

changes

by helping

RT genes,

Discussion Needless to say, the model for carcinogenesis developed here puts utmost of cellular membranes, because the well being of the organism depends which

inhibits

cell division,

for cell division, can initiate

can be affected

maximum

that can

Qualitative

shorten

well as the enzymes

the model

refers

was found

effect

toward

both

of these

tendency

forces adversely

or after being formed

are not made

would

occur

then manifest

possibilities

quantity

without

growing

of new cancer specific

tumors

attempting

of glycoprotein

sugars or other

The

most

to be specific,

probably

recep-

components

by the time GI is brought

itself as the presence rapidly

quantity

if some of the amino

in sufficient

normally.

the end of the GI phase of cell cycle (110).

GI can result at least in less than the required

part of the cell cycle,

to those

which

while being synthesized

can also conceivably

involved

end. This phenomenon is the only variable

either

for glycoproteins

changes

of MI, on the one hand, and the ever present Thus events

the path of no return. adversely

rate of synthesis

Any events

by the effect

by RT, on the other.

the move toward

Membranes

tors.

enhanced

stimulated

importance on the normal state upon the balance between Cl,

as

to an untimely

antigens.

Since Gl

have a shorter

GI. Thus

in a situation

where there are too

many variables.

The most commits perhaps

important after

is committed

some

which

of this paper,

transformed.

to divide.

The model

can be tested

the critical

arginine

If, however,

event

rich f3 histones.

due to defective

Up to that

for adjustment,

point

to resume furnishes

experimentally. during

the right time, when no more proliferation methylate

” of a normal

in the “ transformation

time needed

to continue

cur at this point tion

event

the cell to remain

Methylases

formation

its normal

a precise

According

cell division is needed.

cell into a neoplastic

elimination

quiescent

mechanism

of membrane

about

under

CI cannot

the cell,

beyond

it, the cell

the events

which oc-

in the first sec-

off of RT genes at

to occur when histone

to be formed

RT receptors,

will allow

developed

cell is the turning

This was postulated

were proposed

state;

to the model,

of a normal

cell is the one which

of the cause

methylases

the influence

of CAMP.

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 degrade

which

could

the remaining

not

be synthesized

cGMP and increase

under

these

conditions

is MI, which,

if produced,

would

help

A-cyc activity.

Membranes can also be destroyed by physical agents, such as irradiation and chemical agents such as hydrocarbons, which are excellent fat and lipid solvents. There is a well documented example of myeloid metaplasia (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 Agrobacterium tumafaciens (116). In the animal field Schistosoma haematobium has been shown to cause hematuria,

anemia

are deposited

and in chronic

cases calculus

in the veins of the bladder

(117).

formation

Formation

which

may become

of true stomach

rats a nematode found in the muscles of certain species of cockroaches sarcoma in the upper airway of the dog was shown to follow infection ugh the mechanisms

which

lead to neoplastic

here that it may be due to RT produced The attractiveness ration

after

of this model

generation

any factor

which

without

was not

stances.

The ideas promoted

insights

are gained

Abbreviations RT: nosine phase

lies in its ability involving

utilized

changes.

for the description

of esteoEven tho-

the suggestion

made

be dismissed.

neoplastic

events which are seen in cells gene-

It also does

of the control

experimentally

of metabolic

its eggs

by feeding

(118). A high incidence by Spirocerca lupi (119).

cases are not known,

agent cannot

to describe

genetic

can be tested

on the control

in these

after

was achieved

not

presume

the involvement

of cell cycle under

and the model

is capable

normal

of improvement

of

circumas new

processes.

used:

replication

trigger.

3’-5’ cyclic

diesterase;

changes

by the infectious

malignant,

tumors

MI: Mitotic

monophosphate;

G-PDE:

inhibitor;

A cyc: adenyl

cGMP phosphodiesterase;

of cell cycle;

G2: gap two

division) phase of cell cycle; hamster ovary cells KI line.

CAMP: adenosine

phase

Cap:

cyclase;

GT: glycosyl of cell cycle;

calcium

pump;

3’-5’ cyclic

G cyc: guanyl transferase;

cyclase;

cGMP: gua-

A-PDE:

CAMP phopho-

ST: sialyl transferase;

S: replication

Cl: contact

monophosphate;

phase

inhibition

of cell cycle;

GI:

gap one

M: mitosis

of movement;

CHO-KI:

(cell Chines

Acknowledgments

This work was presented

in part at the X Turkish

Haematology

Society

Meeting

in Ankara,

1975

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