Department of Biochemistry

Federal University Otuoke, Bayelsa State

BCH 411- Seminar in Biochemistry

The Cell Cycle

By

Onwuasoanya Nwike Irenaeus

Matric No: FUO/16/BCH/3928

MARCH 2020

I
 THE CELL CYCLE

By

ONWUASOANYA NWIKE IRENAEUS

FUO/16/BCH/3928

A seminar paper presented to the Department of Biochemistry, Faculty of Science, Federal

University Otuoke, Bayelsa State, Nigeria, in partial fulfilment of the requirements for the
award of Bachelor of Science (B.Sc. Hons) Degree in Biochemistry.

II
 CERTIFICATION

This seminar work entitled “THE CELL CYCLE” was written and presented

by ONWUASOANYA NWIKE IRENAEUS, FUO/16/BCH/3928 to the Department

of Biochemistry, Faculty of Science, Federal University Otuoke, Bayelsa

State, Nigeria.

PROFESSOR UADIA PATRICK DATE

(Seminar Supervisor)

DR. EJOVI OSIOMA

DATE
(Head of Department)

III
 DEDICATION

This work is dedicated to God Almighty

IV
 ACKNOWLEDGEMENTS

I am grateful to my seminar supervisor Prof. P. Uadia for his guidance and advice
towards the completion of this work, the HOD biochemistry department Dr. Ejovi
Osioma, and also the entire biochemistry lecturers; Prof. O. Adeyemi, Prof. C. C.
Onyenekwe, Prof. O. Adeyemi, Dr. E. N Agomuo, Dr. D. O. Kpomah, Dr. A. N.
Okpogba, Dr. C. E. Igwe, Dr. R. U. Nkiruka, Dr. E. S. Agoro, Dr. O. B. Ilesanmi,
Dr. J. T. Johnson, Mr. E. A. Gbodo, Mr. G. C. Ikimi for the knowledge they have
impacted in me.
Thank you all.

V
 Abstract

The cell cycle is the name given to the process by which a cell matures,
synthesizes DNA, and divides to form daughter cells. Thus the cell cycle is a
fundamental process with analogous mechanisms found in all cells, from the most
primitive bacterium to higher animals and plants, from the unicellular to the most
complex multicellular organism. Cells use special proteins and checkpoint
signalling systems to ensure that the cell cycle progresses properly. Checkpoints at
the end of G1 and at the beginning of G2 are designed to assess DNA for damage
before and after S phase. Likewise, a checkpoint during mitosis ensures that the
cell’s spindle fibres are properly aligned in metaphase before the chromosomes are
separated in anaphase. If DNA damage or abnormalities in spindle formation are
detected at these checkpoints, the cell is forced to undergo programmed cell death,
or apoptosis. However, the cell cycle and its checkpoint systems can be sabotaged
by defective proteins or genes that cause malignant transformation of the cell,
which can lead to cancer. For example, mutations in a protein called p53, which
normally detects abnormalities in DNA at the G1 checkpoint, can enable cancer-
causing mutations to bypass this checkpoint and allow the cell to escape apoptosis.

Keywords: Mitosis, Cytokinesis, Karyokinesis, Kinetochores, Mitotic spindle, Cyclins,

Cyclin-dependent Kinases, Centromere, Centrosomes, Checkpoints, Cleavage Furrow.

VI
Table of Contents

CERTIFICATION......................................................................................................................III
DEDICATION.............................................................................................................................IV
ACKNOWLEDGEMENTS.........................................................................................................V
Abstract........................................................................................................................................VI
LIST OF FIGURES.................................................................................................................VIII
LIST OF TABLES......................................................................................................................IX
CHAPTER ONE............................................................................................................................1
1.1 INTRODUCTION.................................................................................................................................1
1.2 HISTORY OF CELL CYCLE.....................................................................................................................2
CHAPTER TWO...........................................................................................................................3
2.1 PHASES..............................................................................................................................................3
2.1.1 INTERPHASE................................................................................................................................3
2.1.2 MITOTIC PHASE...........................................................................................................................6
2.2 PROGRAMMED CELL DEATH............................................................................................................12
Programmed Cell Death versus Accidental Cell Death: Apoptosis versus Necrosis..........................12
CHAPTER THREE.....................................................................................................................13
3.1 REGULATION AND CHECKPOINTS OF THE CELL CYCLE.....................................................................13
CHAPTER FOUR.......................................................................................................................15
4.1 DISCUSSION.....................................................................................................................................15
4.2 CONCLUSION...................................................................................................................................16
REFERENCES............................................................................................................................17

VII
 LIST OF FIGURES

FIGURE 1................................................................................................................................................... 3

FIGURE 2..................................................................................................................................................5

FIGURE 3................................................................................................................................................ 7

FIGURE 4.................................................................................................................................................8

FIGURE 5................................................................................................................................................ 8

FIGURE 6................................................................................................................................................9

FIGURE 7.............................................................................................................................................10

FIGURE 8.............................................................................................................................................. 11

FIGURE 9...............................................................................................................................................12

VIII
LIST OF TABLES

IX
 CHAPTER ONE
1.1 INTRODUCTION

The cell cycle is an ordered series of events involving cell growth and cell division that produces
two new daughter cells. Cells on the path to cell division proceed through a series of precisely
timed and carefully regulated stages of growth, DNA replication, and division that produce two
genetically identical cells.

The cell cycle can be thought of as the life cycle of a cell. In other words, it is the series of
growth and development steps a cell undergoes between its birth, formation by the division of a
mother cell and reproduction, division to make two new daughter cells.

To divide, a cell must complete several important tasks: it must grow, copy its genetic material
(DNA), and physically split into two daughter cells. Cells perform these tasks in an organized,
predictable series of steps that make up the cell cycle. The cell cycle is a cycle, rather than a
linear pathway, because at the end of each go-round, the two daughter cells can start the exact
same process over again from the beginning.

In eukaryotic cells, or cells with a nucleus, the stages of the cell cycle are divided into two major
phases: interphase and the mitotic (M) phase. During interphase, the cell grows and makes a
copy of its DNA. During the mitotic (M) phase, the cell separates its DNA into two sets and
divides its cytoplasm, forming two new cells. (Fleming et al, 1882)

Different cells take different lengths of time to complete the cell cycle. A typical human cell
might take about 24 hours to divide, but fast-cycling mammalian cells, like the ones that line the
intestine, can complete a cycle every 9-10 hours when they're grown in culture. Different types
of cells also split their time between cell cycle phases in different ways. In early frog embryos,
for example, cells spend almost no time in G1 and G2 and instead rapidly cycle between S and M
phases, resulting in the division of one big cell, the zygote, into many smaller cells. (Reece et al,
2014).
1.2 HISTORY OF CELL CYCLE

Walther Flemming, a 19th century professor at the Institute for Anatomy in Kiel, Germany, was
the first to document the details of cellular division. The use of microscopes to study biological
tissues was an emerging technology in Flemming's day, and he was highly regarded as an
innovator in the field.

As a professor at Kiel, Flemming experimented with a technique for using dyes to color the
specimens he wanted to examine under a microscope. Microscopes in the 1870s were not
equipped with electric light sources as they are today, so dying the specimens allowed him to see
them in greater detail. He found aniline dyes particularly useful because different types of tissues
absorbed the dyes at varying intensities depending on their chemistry. The effect was that
different parts of a cell would absorb more dye, in effect "highlighting" them, to reveal structures
and processes that were invisible before.

Flemming used these dyes to study cells. In particular, he was interested in the process of cell
division. He began a series of live observations under the microscope using dyed samples of
animal tissues and found that a particular mass of material inside the nucleus of cells absorbed
the dye quite well. He didn't have a name for it at the time, but later came to call the material
"chromatin," from chroma, the Greek word for color (Zacharias, 2013). Flemming drew pictures
of what he saw under his microscope to illustrate various publications he produced in his
research.

Flemming did many of his experiments with tissue samples from Fire salamanders, a common
species in Northern European forests, because the chromatin in their nuclei was large in
comparison to other available study organisms. After many hours of observation, Flemming
began to see a pattern whereby cells would periodically transition from a resting stage to a period
of frenzied activity that turned one nucleus into two, and then pulled the entire cell apart creating
two identical cells – each with its own complement of chromatin enveloped within its nucleus.

Today we call the process of the nucleus splitting into two nuclei mitosis, and the cell split itself,
cytokinesis. The terms came into use years after Flemming's discovery, but he described the
process fully in his book Zur Kenntniss der Zelle und ihrer Theilungs-Erscheinungen (To the
knowledge of the cell and its phenomena of division) (Flemming, 1878).
 CHAPTER TWO
2.1 PHASES

The cell cycle is a four-stage process in which the cell increases in size (gap 1, or G1, stage),
copies its DNA (synthesis, or S, stage), prepares to divide (gap 2, or G2, stage), and divides
(mitosis, or M, stage). The stages G1, S, and G2 make up interphase, which accounts for the span
between cell divisions. On the basis of the stimulatory and inhibitory messages a cell receives, it
decides whether it should enter the cell cycle and divide. (Rieder et al 2002).

2.1.1 INTERPHASE

Interphase, which appears to the eye to be a resting stage between cell divisions, is actually a
period of diverse activities. Those interphase activities are indispensable in making the next
mitosis possible. Interphase generally lasts at least 12 to 24 hours in mammalian tissue. During
this period, the cell is constantly synthesizing RNA, producing protein and growing in size. By
studying molecular events in cells, scientists have determined that interphase can be divided into
4 steps: Gap 0 (G0), Gap 1 (G1), S (synthesis) phase, Gap 2 (G2).

Figure 1

G0 PHASE:

The G0 phase (referred to the G zero phase) or resting phase is a period in the cell cycle in which
cells exist in a quiescent state. G0 phase is viewed as either an extended G1 phase, where the cell
is neither dividing nor preparing to divide, or a distinct quiescent stage that occurs outside of the
cell cycle. Some types of cells, such as nerve and heart muscle cells, become quiescent when
they reach maturity (i.e., when they are terminally differentiated) but continue to perform their
main functions for the rest of the organism's life. Multinucleated muscle cells that do not
undergo cytokinesis are also often considered to be in the G0 stage. On occasion, a distinction in
terms is made between a G0 cell and a 'quiescent' cell (e.g., heart muscle cells and neurons),
which will never enter the G1 phase, whereas other G0 cells may.
Cells enter the G0 phase from a cell cycle checkpoint in the G1 phase, such as the restriction
point (animal cells) or the start point (yeast). This usually occurs in response to a lack of growth
factors or nutrients. During the G0 phase, the cell cycle machinery is dismantled and cyclins and
cyclin-dependent kinases disappear. Cells then remain in the G0 phase until there is a reason for
them to divide. Some cell types in mature organisms, such as parenchymal cells of the liver and
kidney, enter the G0 phase semi-permanently and can be induced to begin dividing again only
under very specific circumstances. Other types of cells, such as epithelial cells, continue to
divide throughout an organism's life and rarely enter G0.
Although many cells in the G0 phase may die along with the organism, not all cells that enter the
G0 phase are destined to die; this is often simply a consequence of the cell's lacking any
stimulation to re-enter in the cell cycle.
Cellular senescence is distinct from quiescence because it is a state that occurs in response to
DNA damage or degradation that would make a cell's progeny nonviable. Senescence then,
unlike quiescence, is often a biochemical alternative to the self-destruction of such a damaged
cell by apoptosis. Furthermore, quiescence is reversible whereas senescence isn't.

G1 PHASE
The g1 phase, or Gap 1 phase, is the first of four phases of the cell cycle that takes place in
eukaryotic cell division. In this part of interphase, the cell synthesizes mRNA and proteins in
preparation for subsequent steps leading to mitosis. G1 phase ends when the cell moves into the
S phase of interphase. During G1 phase, the cell grows in size and synthesizes mRNA and
proteins (known as histones) that are required for DNA synthesis. Once the required proteins and
growth are complete, the cell enters the next phase of the cell cycle, S phase. The duration of
each phase, including the G1 phase, is different in many different types of cells. In human
somatic cells, the cell cycle lasts about 18 hours, and the G1 phase takes up about 1/3 of that
time. However, in Xenopus embryos, sea urchin embryos, and Drosophila embryos, the G1
phase is barely existent and is defined as the gap, if one exists, between the end of mitosis and
the S phase.

G1 phase and the other sub phases of the cell cycle may be affected by limiting growth factors
such as nutrient supply, temperature, and room for growth. Sufficient nucleotides and amino
acids must be present in order to synthesize mRNA and proteins. Physiological temperatures are
optimal for cell growth. In humans, the normal physiological temperature is around 37 °C (98.6
°F).

G1 phase is particularly important in the cell cycle because it determines whether a cell commits
to division or to leaving the cell cycle. If a cell is signaled to remain undivided, instead of
moving onto the S phase, it will leave the G1 phase and move into a state of dormancy called the
G0 phase. Most nonproliferating vertebrate cells will enter the G0 phase.
During the G1 phase of the cell cycle, each cell makes a key decision: whether to continue
through another cycle and divide or to remain in a nondividing state either temporarily or
permanently. During development of metazoans, cells exit the cell cycle as the first step toward
forming differentiated tissues. In adults, strict regulation of the timing and location of cell
proliferation is critical to avoid cancer. Cells enter G1 phase at the end of a proliferation cycle,
after completing mitosis. To be free to decide whether to proliferate or differentiate, the cell must
inactivate the remnants of the proliferation machinery from the preceding cell cycle. This is
initiated in late M-phase by inactivating cyclin-dependent kinases (Cdks) via proteolytic
destruction of their cyclin subunits. This continues in G1 phase and is accompanied by synthesis
and stabilization of Cdk-inhibitory proteins. The absence of Cdk activity activates a regulatory
network that represses the transcription of many genes that promote cell-cycle progression.
While this repressive network is active, the cell cannot proceed through the cell cycle. The
repression can be switched off if the cell is stimulated by specific signals from the surrounding
medium, extracellular matrix, and other cells. If these signals are diffusible substances, they are
known as mitogens. Mitogens can trigger another round of DNA replication and mitosis, but
first, the cell must pass a major decision point in G1 called the restriction point.

S PHASE

To produce two similar daughter cells, the complete DNA instructions in the cell must be
duplicated. DNA replication occurs during this S (synthesis) phase. During S phase, which
follows G1 phase, all of the chromosomes are replicated. Following replication, each
chromosome now consists of two sister chromatids. Thus, the amount of DNA in the cell has
effectively doubled, even though the ploidy, or chromosome count, of the cell remains at 2n.
(Karsenti et al, 2011). Note: Chromosomes double their number of chromatids post replication
but the nuclei remains diploid as the number of centromeres and chromosomes remains
unchanged. Hence, the number of chromosomes in the nucleus, which determines the ploidy,
remains unchanged from the beginning to the end of the S phase.

Figure 2

RNA transcription and protein production are very low during this phase. Since there are a bunch
of proteins and enzymes working to replicate the DNA, it is hard to transcribe RNA and make
proteins from it. However, histones, the proteins that hold DNA together, are created during this
phase. They are necessary for chromosome formation.

Normally, a cell contains all of its DNA in a loose mass called chromatin inside of the nucleus.
This open, loose structure of DNA makes it easily accessible by all of the enzymes and it stays in
this form during the S phase.

During this phase, the cell is said to contain 2n chromosomes. Even though the DNA is not
formed into chromosomes yet, it contains twice the material that goes into the chromosomes. The
n is the number of chromosomes typically in the cell. If a cell normally has n=7 chromosomes,
then during S phase it has 2n chromosomes (14).

G2 PHASE
In the G2 phase, the cell replenishes its energy stores and synthesizes proteins necessary for
chromosome manipulation. Some cell organelles are duplicated, and the cytoskeleton is
dismantled to provide resources for the mitotic phase. There may be additional cell growth
during G2. The final preparations for the mitotic phase must be completed before the cell is able
to enter the first stage of mitosis.

Curiously, G2 phase is not a necessary part of the cell cycle, as some cell types (particularly
young Xenopus embryos [1] and some cancers [2]) proceed directly from DNA replication to
mitosis. Though much is known about the genetic network which regulates G2 phase and
subsequent entry into mitosis, there is still much to be discovered concerning its significance and
regulation, particularly in regards to cancer. One hypothesis is that the growth in G2 phase is
regulated as a method of cell size control. (Linksay, 1977).

2.1.2 MITOTIC PHASE

MITOSIS
Mitosis is a form of eukaryotic cell division that produces two daughter cells with the same
genetic component as the parent cell. Chromosomes replicated during the S phase are divided in
such a way as to ensure that each daughter cell receives a copy of every chromosome. In actively
dividing animal cells, the whole process takes about one hour. The replicated chromosomes are
attached to a 'mitotic apparatus' that aligns them and then separates the sister chromatids to
produce an even partitioning of the genetic material. This separation of the genetic material in a
mitotic nuclear division (or karyokinesis) is followed by a separation of the cell cytoplasm in a
cellular division (or cytokinesis) to produce two daughter cells.
Mitosis is an ancient process, and a number of variations emerged during eukaryotic evolution.
Many singlecelled eukaryotes, including yeast and slime molds, undergo a closed mitosis, in
which spindle formation and chromosome segregation occur within an intact nuclear envelope to
which the spindle poles are anchored.

In some single-celled organisms mitosis forms the basis of asexual reproduction. In diploid
multicellular organisms sexual reproduction involves the fusion of two haploid gametes to
produce a diploid zygote. Mitotic divisions of the zygote and daughter cells are then responsible
for the subsequent growth and development of the organism. In the adult organism, mitosis plays
a role in cell replacement, wound healing and tumor formation. Mitosis, although a continuous
process, is conventionally divided into five stages: prophase, prometaphase, metaphase, anaphase
and telophase.

Figure 3

PROPHASE

Prophase occupies over half of mitosis. The nuclear membrane breaks down to form a number of
small vesicles and the nucleolus disintegrates. A structure known as the centrosome duplicates
itself to form two daughter centrosomes that migrate to opposite ends of the cell. The
centrosomes organise the production of microtubules that form the spindle fibres that constitute
the mitotic spindle. The chromosomes condense into compact structures. Each replicated
chromosome can now be seen to consist of two identical chromatids (or sister chromatids) held
together by a structure known as the centromere. (Kapoor et al, 2011)

EARLY PROPHASE: In early prophase, the cell starts to break down some structures and build
others up, setting the stage for division of the chromosomes. The chromosomes start to condense
(making them easier to pull apart later on). (Openstax, 2016)
 Figure 4

The mitotic spindle begins to form. The spindle is a structure made of microtubules, strong fibers
that are part of the cell’s “skeleton.” Its job is to organize the chromosomes and move them
around during mitosis. The spindle grows between the centrosomes as they move apart. The
nucleolus, a part of the nucleus where ribosomes are made, disappears. This is a sign that the
nucleus is getting ready to break down.

PROMETAPHASE: The chromosomes, led by their centromeres, migrate to the equatorial plane
in the mid-line of the cell - at right-angles to the axis formed by the centrosomes. This region of
the mitotic spindle is known as the metaphase plate. The spindle fibres bind to a structure
associated with the centromere of each chromosome called a kinetochore. (Kai et al, 2009).
Individual spindle fibres bind to a kinetochore structure on each side of the centromere. The
chromosomes continue to condense.

Figure 5

METAPHASE

A stage of mitosis in the eukaryotic cell cycle in which chromosomes are at their second-most
condensed and coiled stage (they are at their most condensed in anaphase).[1] These
chromosomes, carrying genetic information, align in the equator of the cell before being
separated into each of the two daughter cells. Metaphase accounts for approximately 4% of the
cell cycle's duration. Preceded by events in prometaphase and followed by anaphase,
microtubules formed in prophase have already found and attached themselves to kinetochores in
metaphase. In metaphase, the centromeres of the chromosomes convene themselves on the
metaphase plate (or equatorial plate), [2] an imaginary line that is equidistant from the two
centrosome poles. This even alignment is due to the counterbalance of the pulling powers
generated by the opposing kinetochore microtubules,(Skibbens et al, 1993) analogous to a tug-
of-war between two people of equal strength, ending with the destruction of B cyclin.[4] In
certain types of cells, chromosomes do not line up at the metaphase plate and instead move back
and forth between the poles randomly, only roughly lining up along the middle line.[] Early
events of metaphase can coincide with the later events of prometaphase, as chromosomes with
connected kinetochores will start the events of metaphase individually before other
chromosomes with unconnected kinetochores that are still lingering in the events of
prometaphase.[]

Figure 6

One of the cell cycle checkpoints occurs during prometaphase and metaphase. Only after all
chromosomes have become aligned at the metaphase plate, when every kinetochore is properly
attached to a bundle of microtubules, does the cell enter anaphase. It is thought that unattached or
improperly attached kinetochores generate a signal to prevent premature progression to
anaphase, even if most of kinetochores have been attached and most of the chromosomes have
been aligned. Such a signal creates the mitotic spindle checkpoint. This would be accomplished
by regulation of the anaphase-promoting complex, securin, and separase.

ANAPHASE

In anaphase, the sister chromatids separate from each other and are pulled towards opposite ends
of the cell. The protein that holds the sister chromatids together is broken down, allowing them
to separate. Each is now its own chromosome. The chromosomes of each pair are pulled towards
opposite ends of the cell. Microtubules not attached to chromosomes elongate and push apart,
separating the poles and making the cell longer. (Albert et al, 2002).

Anaphase starts when the anaphase promoting complex marks an inhibitory chaperone called
securin for destruction by ubiquinylating it. Securin is a protein which inhibits a protease known
as separase. The destruction of securin unleashes separase which then breaks down cohesin, a
protein responsible for holding sister chromatids together. [2]
 Figure 7

At this point, three subclasses of microtubule unique to mitosis are involved in creating the
forces necessary to separate the chromatids: kinetochore microtubules, interpolar microtubules,
and astral microtubules. The centromeres are split, and the sister chromatids are pulled toward
the poles by kinetochore microtubules. They take on a V-shape or Y-shape as they are pulled to
either pole. While the chromosomes are drawn to each side of the cell, interpolar microtubules
and astral microtubules generate forces that stretch the cell into an oval. [3]

TELOPHASE

The main events of telophase include a reappearance and enlargement of the nucleolus,
enlargement of the daughter nuclei to their interphase size, de-condensation of the chromatin
resulting in a brighter appearance of the nuclei with phase-contrast optics, and a period of rapid,
post-mitotic nuclear migration during which the daughter nuclei become positioned prior to
septum formation (Aist, 1969, 1995). Although the natural breaking of the spindle is used to
define the onset of telophase (Bayles et al., 1993), telophase events involving the nucleolus, the
chromatin, and nuclear size frequently begin moments before the spindle breaks. Thus,
there is sometimes overlap between the anaphase and telophase stages regarding
the behaviour of the various nuclear components. This is one reason why it is helpful to use
only one of several available criteria, (i.e., spindle breakdown) to define the starting point for
telophase. The other reason is that the daughter nuclei are not truly independent of each other
until spindle breakdown; therefore, technically, the nucleus is still dividing. The phosphorylation
of the protein targets of M-Cdks (Mitotic Cyclin-dependent Kinases) drives spindle assembly,
chromosome condensation and nuclear envelope breakdown in early mitosis. The
dephosphorylation of these same substrates drives spindle disassembly, chromosome
decondensation and the reformation of daughter nuclei in telophase. Establishing a degree of
dephosphorylation permissive to telophase events requires both the inactivation of Cdks and the
activation of phosphatases.
 Figure 8

CYTOKINESIS

Cytokinesis, or “cell motion,” is the second main stage of the mitotic phase during which cell
division is completed via the physical separation of the cytoplasmic components into two
daughter cells. Division is not complete until the cell components have been apportioned and
completely separated into the two daughter cells. Although the stages of mitosis are similar for
most eukaryotes, the process of cytokinesis is quite different for eukaryotes that have cell walls,
such as plant cells.

In cells such as animal cells that lack cell walls, cytokinesis follows the onset of anaphase. A
contractile ring composed of actin filaments forms just inside the plasma membrane at the former
metaphase plate. The actin filaments pull the equator of the cell inward, forming a fissure. This
fissure, or “crack,” is called the cleavage furrow. The furrow deepens as the actin ring contracts,
and eventually the membrane is cleaved in two. (Blo et al, 2005).

In plant cells, a new cell wall must form between the daughter cells. During interphase, the Golgi
apparatus accumulates enzymes, structural proteins, and glucose molecules prior to breaking into
vesicles and dispersing throughout the dividing cell. During telophase, these Golgi vesicles are
transported on microtubules to form a phragmoplast (a vesicular structure) at the metaphase
plate. There, the vesicles fuse and coalesce from the center toward the cell walls; this structure is
called a cell plate. As more vesicles fuse, the cell plate enlarges until it merges with the cell walls
at the periphery of the cell. Enzymes use the glucose that has accumulated between the
membrane layers to build a new cell wall. The Golgi membranes become parts of the plasma
membrane on either side of the new cell wall.
 Figure 9

2.2 PROGRAMMED CELL DEATH

The ability to undergo programmed cell death is a built-in latent capacity in most cells of
multicellular organisms. Cell death is important for embryonic development, maintenance of
tissue homeostasis, establishment of immune self-tolerance, killing by immune effector cells, and
regulation of cell viability by hormones and growth factors. Both extrinsic signals and internal
imbalances can lead cells to kill themselves. Furthermore, many metazoan cells will die if they
fail to receive survival signals from other cells. Abnormalities of the cell death program
contribute to several important diseases, including cancer, Alzheimer disease, and AIDS. Cell
death programs are ancient: much of the current network was present in the last eumetazoan
common ancestor (Cell Biology, 2017).

Programmed Cell Death versus Accidental Cell Death: Apoptosis versus Necrosis
Although cells die in many ways, it is useful to focus on two poles of this spectrum: apoptosis
and necrosis. Apoptosis, the most widely studied pathway for programmed cell death, is cellular
suicide resulting from activation of a dedicated intracellular program. At the other end of the
spectrum necrosis, also called accidental cell death, occurs when cells sustain a structural or
chemical insult that causes the cells to swell and undergo membrane lysis. Examples of such
insults include extremes of temperature and physical trauma. Cells can also initiate active
programmed necrosis in response to certain stimuli, particularly when induction of apoptosis is
inhibited. Programmed necrosis looks morphologically like accidental cell death. A third
pathway leading to cell death involves autophagy. Although usually regarded as a protective
response to starvation, autophagy has been implicated in certain examples of cell death,
particularly during development. Necrosis corresponds to what most of us naively imagine cell
death would be like. Owing to lack of cellular homeostasis, water rushes into the dying cell,
causing it to swell until the plasma and organelle membranes burst.(Cell Biology, 2017).
 CHAPTER THREE
3.1 REGULATION AND CHECKPOINTS OF THE CELL CYCLE
The checkpoints are evolutionarily conserved surveillance mechanisms controlling the order and
timing of cell cycle transitions. They are organized as signal transduction cascades blocking or
slowing down cell cycle progression at specific stages. Checkpoints are triggered by sensor
proteins detecting, directly or indirectly, cell cycle perturbations and transmitting the signal,
through the action of protein kinases, to effector proteins that stop cell cycle progression until the
signal activating the checkpoint has been turned off. These mechanisms have been highly
conserved during evolution, and checkpoint defects result in genome instability, which is
frequently associated to tumour development. The checkpoint controls are elicited through
molecular events regulating their activation, maintenance, and inactivation resulting,
respectively, in cell cycle arrest, maintenance of the arrest for a certain time and recovery of cell
cycle progression. These surveillance mechanisms can be divided into intrinsic regulatory
pathways, ensuring the orderly progression of cell cycle events under physiological conditions,
and extrinsic pathways that are activated in response to specific clues, such as damage to DNA
or cellular structures. The intrinsic checkpoints act by controlling the activity of cell cycle
dependent kinases (CDKs) mainly at the G1/S boundary and at the metaphase to anaphase
transition in mitosis; such mechanisms are described in other entries of the encyclopaedia.

DNA Damage Checkpoints

The DNA damage checkpoint is required for the efficient response to genotoxic stress. The
checkpoint is activated when lesions in the DNA are detected and the mechanisms involved
differ slightly at various cell cycle phases. DNA damage during the G1 phase activates the G1/S
checkpoint preventing entry into S phase. The presence of DNA lesions while cells replicate
their genome slows down the kinetics of DNA replication (intra-S checkpoint), and if the
chromosomes are damaged in G2, the activation of the G2/M checkpoint avoids chromosome
segregation before repair. Precise and complete DNA replication in every cell cycle and repair of
DNA lesions are critical for the maintenance of genetic stability; failures in these processes
reduce cell survival and lead to cancer susceptibility. Cell cycle arrest is not the only final
outcome of the DNA damage checkpoint response; indeed, it has been demonstrated that
checkpoint activation regulates the choice of recombination pathways, influences transcription of
DNA repair genes, stabilizes stalled replication forks and, in multicellular eukaryotes, it may
promote apoptosis when the damage is irreparable.

The Spindle Assembly Checkpoint

Chromosome segregation at mitosis is controlled by two surveillance mechanisms: the spindle

assembly and the spindle positioning checkpoints. Accurate segregation requires bipolar
attachment of sister chromatids to the mitotic spindle, which is mediated by a proper connection
between kinetochores and spindle microtubules. Kinetochore capture and microtubules bi-
orientation are stochastic processes taking a variable amount of time to complete. During that
time individual chromosomes may be detached from the microtubules or be connected only to
one spindle pole. The spindle assembly checkpoint (SAC) delays the metaphase to anaphase
transition until the sister chromatids are properly attached to the spindle in a bipolar orientation.
In budding yeast, cell cycle arrest at the G2/M transition is mediated by inhibition of the CDC20-
anaphase promoting complex (APC) ubiquitin ligase, thus preventing proteolysis of the securin
PDS1 until complete bi-orientation is achieved Checkpoint proteins (MAD1, MAD2, BUB1,
BUBR1, BUB3, and MPS1) all accumulate at unattached kinetochores and form various
complexes, many of which can inhibit the APC (Fang et al. 1998). APC is a multiprotein
complex that targets several proteins for degradation during mitosis through the associated
specificity factor CDC20. Securin and cyclins are ubiquitylated by CDC20-APC; therefore, to
delay anaphase onset in the presence of spindle defects, the checkpoint must block CDC20-APC
mediated PDS1 degradation. Experimental evidence suggests that in response to spindle defects,
MAD2 exchanges from a MAD1/MAD2 complex to a CDC20/MAD2 complex sequestering
CDC20 away from the APC and blocking PDS1 degradation. In S. cerevisiae, spindle
misorientation is detected by the spindle positioning checkpoint (SPOC) which prevents mitotic
exit. The target of this control is the mitosis exit network (MEN), and more specifically the
activation of the TEM1 GTPase (Adames et al. 2001). TEM1 cycles between GDP- and GTP-
bound states, regulated by the putative guanine nucleotide exchange factor (GEF) LTE1 and the
two-component GTPase activating protein (GAP) BFA1/BUB2. The last one recruits TEM1 to
the bud-directed spindle pole, where TEM1 is kept inactive until the pole crosses the neck into
the bud. GTP-TEM1 then binds to the protein kinase CDC15, which phosphorylates and
activates the protein kinase DBF2. MOB1 binds to DBF2 and, in a poorly understood manner,
MOB1/DBF2 stimulates the release of the CDC14 phosphatase from the nucleolus and
contributes to cytokinesis. CDC14 dephosphorylates CDH1, leading to the activation of CDH1-
APC complex, which triggers cyclin degradation and exit from mitosis(Lew and Burke 2003).
 CHAPTER FOUR
4.1 DISCUSSION

Research on the animal cell cycle has entered a new phase during the last few years. The
identification and purification of growth factors have made possible studies on the interaction of
these factors with cell membrane receptors and will eventually lead to elucidation of the steps
between absorption of growth factors and their ultimate effects on activating processes within the
cell. These investigations will fuse with those on membranal and submembranal structure and
function, as well as with studies on the role of intracellular "second messengers" such as cyclic
nucleotides. The Go and G1 parts of the cell cycle are still largely "gaps" with regard to our
knowledge of biochemical events which are either necessary or sufficient for the initiation of
DNA synthesis. That the transition from Go to S requires prolonged exposure to growth factors
argues against the sufficiency of an early triggering event. Serum has been the substance most
used to stimulate quiescent cells. The great complexity of serum leaves open the possibility that
some of the serum-induced biochemical changes may be unrelated to the initiation of DNA
synthesis. The use of purified growth factors to stimulate cells will help to define more clearly
the biochemical changes directly related to growth stimulation. Attempts are underway to
identify proteins, RNAs, smaller molecules, and structures that are directly involved in progress
through the cycle and to determine the ways in which their synthesis is regulated. Ideas about the
nature of quiescence have been in flux, due in part to the introduction of the probabilistic model
and the restriction point hypothesis. The proposal that normal cells enter quiescence if they
cannot accomplish a specific regulatory event in G2 and that transformed cells have escaped in
whole or in part from the need to accomplish this event, or can accomplish it more easily,
provides the basis for further experimentation on the nature of cancer. Elucidation of the timing
and the biochemical nature of the restriction event is on the horizon. Changes in the nucleus and
chromatin occur throughout the cell cycle. Their causal relationships to other events in the cell
cycle and to progress through the cycle remain to be discovered. We will have to ask questions
regarding the importance of the transcription of RNA and its subsequent processing and transport
into the cytoplasm, nuclear preparation for DNA synthesis during G2 the event directly
responsible for initiation of DNA synthesis, and the orderly progression of replication of
different parts of the genome. G2 is also almost devoid of landmarks. Inhibitors are known to be
more effective at certain times in G2 than at other times in the cycle. Our ability to obtain cell
cycle mutants is still limited. Each new mutant, however, should lead to further information
about necessary cycle steps. In this regard, the more developed studies on yeast cell cycle
mutants should suggest regulatory mechanisms that might also be operative in higher cells.
4.2 CONCLUSION

Recent advances in our understanding of the cell cycle have revealed numerous regulatory
processes that ensure the order of events in the cell cycle and integrate repair processes with cell
cycle progression. Defects in these cell cycle controls can render the normal responses to damage
ineffective and can lead to genomic instability and progression to malignancy. As our knowledge
of these processes increases, we will be able to use molecular and cellular assays to assess the
cell cycle controls missing in specific tumors. This characterization may dictate the choice and
schedule of agents to be used in therapy. New compounds are likely to be developed that take
advantage of the differences between cell cycle control in normal and cancer cells to maximize
therapeutic effectiveness. Many of these new agents may be biological modifiers, rather than
nonselective cytotoxic agents, that influence how cells respond to cytotoxic agents in terms of
cell cycle perturbations and cell death pathways. For some cancers, the ultimate therapy-
prevention strategies-may also be devised on the basis of this knowledge.
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