Assignment on Cell cycles and its regulation. Cell death– events, regulators, intrinsic and extrinsic pathways of apoptosis. Necrosis and autophagy.

1.  The cell cycle is the series of events in which
cellular components are doubled, and then
accurately segregated into daughter cells.
4/30/2018 1
Phases of cell cycle
Interphase
 G1
 S
 G2
Mitotic phase
 karyokinesis
 Cytokinesis

2.  Cell cycle checkpoints are surveillance mechanisms
that monitor the order, integrity, and fidelity of the
major events of the cell cycle.
 These include growth to the appropriate cell size, the
replication and integrity of the chromosomes, and their
accurate segregation at mitosis.
4/30/2018 2

3. Cell cycle
M
phase
G1
phase
S
phase
G2
phase
4/30/2018 3
Mitosis
cytokinesis
Mitotic
phase
interphase
Nutrition
Growth factors
Cell growth
Cell size
Cell growth
DNA Synthesis
Two
daughter
cells
formation

4.  G1 phase. Metabolic changes prepare the cell
for division. At a certain point - the
restriction point - the cell is committed to
division and moves into the S phase.
 S phase. DNA synthesis replicates the genetic
material. Each chromosome now consists of
two sister chromatids.
 G2 phase. Metabolic changes assemble the
cytoplasmic materials necessary for mitosis
and cytokinesis.
.
4/30/2018 4

5.  M phase. A nuclear division (mitosis) followed
by a cell division (cytokinesis).
4/30/2018 5

6.  It is a form of eukaryotic cell division that
produces two daughter cells with the same
genetic component as the parent cell.
 Mitosis, although a continuous process, is
conventionally divided into five stages:
prophase, prometaphase, metaphase,
anaphase and telophase
4/30/2018 6

7.  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.
 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.
4/30/2018 7

8. 4/30/2018 8

9. 4/30/2018 9

10. 4/30/2018 10
Metaphase
Metaphase is characterized by
the "metaphase plate".
This is a mid-point region within
the cell that is formed/defined by
the centromeres of the chromatid
pairs aligning along the
microtubules at the centre of the
miotic spindle

11. 4/30/2018 11

12.  Telophase begins after the chromosomal
movement stops.
 The identical sets of chromosomes - which
are by this stage at opposite poles of the cell,
uncoil and revert to the long, thin, thread-
like chromatin form.
 A new nuclear envelope forms around each
chromatin mass.
 Nucleoli appear.
 Eventually the miotic spindle breaks-up
4/30/2018 12

13. 4/30/2018 13

14.  The final cellular division to form two new
cells.
 In plants a cell plate forms along the line of
the metaphase plate; in animals there is a
constriction of the cytoplasm.
 The cell then enters interphase - the interval
between mitotic divisions
4/30/2018 14

15. 4/30/2018 15

16. 4/30/2018 16

17.  Meiosis is the form of eukaryotic cell division
that produces haploid sex cells or gametes
from diploid cells (which contain two copies
of each chromosome).
 The process takes the form of one DNA
replication followed by two successive nuclear
and cellular divisions (Meiosis I and Meiosis
II).
 As in mitosis, meiosis is preceded by a
process of DNA replication that converts each
chromosome into two sister chromatids
4/30/2018 17

18.  In Meiosis I a special cell division reduces the
cell from diploid to haploid
 The homologous chromosomes pair and
exchange DNA to form recombinant
chromosomes. Prophase I is divided into five
phases
4/30/2018 18

19.  Leptotene: chromosomes start to condense.
 Zygotene: homologous chromosomes
become closely associated (synapsis) to form
pairs of chromosomes (bivalents) consisting
of four chromatids (tetrads).
 Pachytene: crossing over between pairs of
homologous chromosomes to form chiasmata
(sing. chiasma).
 Diplotene: homologous chromosomes start to
separate but remain attached by chiasmata.
 Diakinesis: homologous chromosomes
continue to separate, and chiasmata move to
the ends of the chromosomes
4/30/2018 19

20.  Spindle apparatus formed, and chromosomes
attached to spindle fibres by kinetochores
4/30/2018 20

21.  Homologous pairs of chromosomes
(bivalents) arranged as a double row along
the metaphase plate. (This is a source of
genetic variation through random
assortment, as the paternal and maternal
chromosomes in a homologous pair are
similar but not identical. The number of
possible arrangements is 2n, where n is the
number of chromosomes in a haploid set.
Human beings have 23 different
chromosomes, so the number of possible
combinations is 223, which is over 8 million.
4/30/2018 21

22.  Anaphase I The homologous chromosomes in
each bivalent are separated and move to the
opposite poles of the cell
 Telophase I The chromosomes become
diffuse and the nuclear membrane reforms
4/30/2018 22

23. 4/30/2018 23

24.  Meiosis II separates each chromosome into
two chromatids .
 The events of Meiosis II are analogous to
those of a mitotic division, although the
number of chromosomes involved has been
halved.
4/30/2018 24

25. 4/30/2018 25

26.  Cell division, cell differentiation and cell death are
the three principal physiological processes that
regulate tissue homoeostasis in multicellular
organisms
 The importance of dysregulated cell cycle
progression and cell death in the pathogenesis of
major diseases, such as cancer,
ischemia/reperfusion injury, atherosclerosis,
infection, inflammation and neurological disorders,
is now well established
4/30/2018 26

27. Cell cycle activators
Cyclins
Cycle dependent kinases
Maturation promoting factor(MPF)
The anaphase promoting
complexcyclosome(APCC)
Cell cycle inhibitors
P53 Gene
P21 Gene
Rb gene
ATM
ATR
4/30/2018 27
These checkpoint signals stop the cell cycle transitions either by inhibiting
the activator or activating the inhibitor

28.  The central machines that drive cell cycle
progression are the cyclin-dependent kinases
(CDKs).
 These are serine/threonine protein kinases that
phosphorylate key substrates to promote DNA
synthesis and mitotic progression
4/30/2018 28
CDK1 and CDK2
• Binds to cyclins A, B, D, E.
CDK4 and CDK6
• Binds to cyclin D

29. G1 phase
D-type cyclins and CDK4 or CDK6
S phase
cyclin E–CDK2
G2 phase
cyclin A–CDK2 and cyclin A–CDK1
M phase
CDK1–cyclin B, A
4/30/2018 29

30. 4/30/2018 30

31. check point
mechanisms
P53
independent(ATM,
ATR) at chk12
P53 dependent
4/30/2018 31
Genome
insult to
cell
Yes No
Activates p53 and
arrest the cell cycle
Deactivates and
continuous cell
cycle

32. 4/30/2018 32

33. 4/30/2018 33

34. 4/30/2018 34
Cell cycle progression is further regulated by two classes of cell cycle
inhibitors
The INK4 proteins
including p16
(INK4a) and p15
(INK4b)
The Cip/Kip
family including
p21, p27 and
p57.
Specific cyclin–CDK complexes and cell
cycle at specific points

35. 4/30/2018 35

36.  A checkpoint is one of several points in the
eukaryotic cell cycle at which the progression
of a cell to the next stage in the cycle can be
halted until conditions are favorable (e.g. the
DNA is repaired).
 These checkpoints occur near the end of G1,
at the G2/M transition, and during metaphase
4/30/2018 36

37. G1 phase check points
G2 phase check point
M phase check point
4/30/2018 37

38.  G1 be divided into 2 portions: G1-pm for G1-postmitotic, and G1-ps for
G1-pre-S
 Whereas G1-pm is relatively constant, G1-ps is variable, and it is this
variability in the duration of G1-ps that contributes to much of the
confusion.
4/30/2018 38

39. 4/30/2018 39

40.  The G2 checkpoint is an intricate signaling network
that regulates the progression of G2 to mitosis (M)
 A node-based model of G2 checkpoint regulation, in
which the action of the central CDK1–cyclin B1 node is
determined by the concerted but opposing activities
of the Wee1 and cell division control protein 25C
(CDC25C) nodes
4/30/2018 40

41. 4/30/2018 41

42.  During mitosis and meiosis, the spindle assembly checkpoint acts
to maintain genome stability by delaying cell division until
accurate chromosome segregation can be guaranteed.
 Accuracy requires that chromosomes become correctly attached
to the microtubule spindle apparatus via their kinetochores.
 When not correctly attached to the spindle, kinetochores activate
the spindle assembly checkpoint network, which in turn blocks
cell cycle progression.
 Once all kinetochores become stably attached to the spindle, the
checkpoint is inactivated, which alleviates the cell cycle block and
thus allows chromosome segregation and cell division to proceed.
4/30/2018 42

43.  The SAC is the most important mechanism of the mitotic
phase, and it ensures that anaphase will not occur until the
chromosomes are correctly aligned at the equatorial plate
 In this sense, cell cycle regulators such as the CDK1-cyclin B
complex are important components of SAC
4/30/2018 43

44.  Mitotic phase targeting disturbs mitosis in tumor cells, triggers the
spindle assembly checkpoint and frequently results in cell death
 The first antimitotics to enter clinical trials aimed to target tubulin.
Although these drugs improved the treatment of certain cancers,
and many anti-microtubule compounds are already approved for
clinical use, severe adverse events such as neuropathies were
observed
 Since then, efforts have been focused on the development of
drugs that also target kinases, motor proteins and multi-protein
complexes involved in mitosis
4/30/2018 44

45. S. no protein Targeting agents
1 AURKA and AURKB Danusertib, AT9283, Barasertib,
Alisertib, ENMD-2076, PF-03814735
2 CDK1 P276-00, Terameprocol, Seliclib,
Dinaciclib
3 Tubulin Vinflunine, ABT-751, Tesetaxel,
Patupilone, Sagopilone, Vintafolide,
TPI 287
4 EG5 Ispinesib, Filanesib, MK-0731,
SB743921
5 26S Proteasome Delanzomib
6 PLK1 Volasertib, GSK 461364
7 CENP-E GSK-923295, Lonafarnib
4/30/2018 45

46. 4/30/2018 46
• The abnormal expression of cell cycle regulators (activators
and inhibitors) can cause alteration of cell division leads to
different types of cancer
• As we known checkpoints plays a important role in cell cycle
progression through all the phases, it is novel target to treat
many of cancers

47.  Cell death is the event of a biological cell
ceasing to carry out its functions.
Two types
 Programmed cell death (PCD)
 Necrosis – killing – non physiological death

48.  Programmed cell death is cell death mediated
by an intracellular program
 Two types:-
 Apoptosis or Type I cell-death (major
importance)
 Autophagy or Type II cell-death (minor
importance)

49.  APOPTOSIS – It is a natural process, in which
a suicide program is activated within the cell
 Fragmentation of DNA occurs
 Cell shrinkage occurs
 It is energy dependent process
 Apoptotic body forms
 DNA cleavage occurs

50.  Apoptosis is voluntary – it is an alternative to
G0
 However apoptosis is absolutely irreversible

52.  Two different pathways are involved
 Intrinsic pathway or mitochondria mediated
death pathway
In this pathway the death inducing stimuli are
originated inside the target cell

53.  Severe genetic damage
 Lack of oxygen
 Presence of viral proteins
 High concentrations of cytosolic Ca++
 Severe oxidatative stress

54.  The intrinsic pathway is fascilated bcl2 family
proteins
which are characterized of one or more BH
Domain (bcl-2 Homology Domain)
 Bcl-2 family proteins were classified into three
categories
 Pro apoptotic Bcl-2 proteins for ex. Bax and Bak
 Anti apoptotic Bcl-2 proteins for ex. Bcl-2, Bcl-
xL
 BH3 only proteins

Caspases-
Caspase are a family of protease enzymes
playing essential roles in programmed cell death

55.  Retinoblastoma protein
 P53
 E2F

56.  When cell lost balance between Pro apoptotic and
anti apoptotic factors Bax is activated
 Bax undergo conformational change and gets
inserted to outer mitochondrial membrane
 It creates pore on outer mitochondrial membrane
which results in MOMP (mitochondrial outer
membrane permeation)
 Cytochrome C is released through pores
 MOMP is also accelerated by increased calcium
level in cytoplasm
 SMAC (secondary mitochondria derived activator
of caspases) is released from mitochondria

57.  SMAC binds with all anti apoptotic factors and
inactivate them
 In cytoplasm cytochrome c binds with Apaf-1
(apoptotic protease activating factor) and pro
caspase9 in an ATP dependent manner to form
multi subunit complex
 The multi subunit complex form apoptosome
 Caspase9 is the initiator caspase which activates
the executioner caspase such as caspase 3 and 7
 Activated caspase3 and caspase7 cleave its target
molecule in cell thus apototic cell death occur

59.  The extrinsic signaling pathway leading to apoptosis involves
transmembrane death receptors that are members of the tumor
necrosis factor (TNF) receptor gene superfamily
 . Members of this receptor family bind to extrinsic ligands and
transduce intracellular signals that ultimately result in the
destruction of the cell.
 The most well characterized ligands of these receptors are FasL,
TNF-alpha, Apo3L, and Apo2L. Corresponding receptors are
FasR, TNFR1, DR3, and DR4/DR5, respectively.
 The binding of ligand to the receptors on the target cell triggers
receptor clustering on the cell surface. Which recruits the
adaptor proteins on the cytoplasmic site of the receptors,
forming death inducing signalling complex (DISC).

60.  Formation of DISC brings procaspase
molecules close to one another, which
facilitates their autocatalytic activation and
release into the cytoplasm where they
activate caspase cascade.
 Caspase8 is activated which is a initiator
caspase which further activates caspase3/6/7
which are executioar caspase
 Once activated, the caspases affect several
cellular functions as part of a process that
results in the death of the cells.

62.  Active caspase-8 also mediates the cleavage of
proapoptotic protein, BID which subsequently releases
mitochondrial proapoptotic factors linking the two
pathways.
 In case of intrinsic pathway, stress signal causes the
binding of cytoplasmic proteins, BAX and BID to the outer
membrane of mitochondria. Another mitochondrial
protein BAK interacts with BAX and BID causing release of
cytochrome c into the cytosol.
 This binds to Apaf-1 which then forms apotosome that
triggers the activation of procaspase-9.
 Activated caspase-9 further initiates the caspase cascade
leading to apoptosis.
 Tumor suppressor p53 protein is a sensor of cellular
stress and play a vital role in initiating apoptosis by
transcriptionally activating proapoptotic proteins BID and
BAX

64.  Autophagy is an intracellular degradation
system that delivers cytoplasmic constituents
to the lysosome.
 It is a normal process by which eukaryotic
cells break down out-dated and damaged
cellular organelles and proteins to be
replaced with new ones.
 It is also a survival mechanism providing
cells with energy and substrates for cellular
processes in times of stress and starvation.

65. Autophagy is a multi-step process involving
initiation, formation of autophagosomes
(vesicles that capture and deliver cytoplasmic
material to lysosomes for digestion),
maturation, and degradation.
4/30/2018 65

67.  Recent studies have clearly demonstrated
that autophagy has a greater variety of
physiological and pathophysiological roles
than expected, such as starvation adaptation,
intracellular protein and organelle clearance,
development, anti-aging, elimination of
microorganisms, cell death, tumor
suppression, and antigen presentation

68.  Necrosis is a form of cell injury which results in the
premature death of cells in living tissue by autolysis.
 Necrosis is caused by external factors to the cell or
tissue, such as infection, toxins, or trauma which
result in the unregulated digestion of cell
components.
 Cause of Necrosis
 Ischemia
 Physical agent
 Chemical agent
 Immunological injury

69.  Denaturation of intracellular proteins
 Enzymatic digestion of the cell

70.  Coagulative necrosis
 Liquefative necrosis
 Caseeous necrosis
 Fat necrosis
 Fibrnoid necrosis

72.  Lewin's CELLS 3rd Edition by George
Plopper, David Sharp , Eric Sikorski published
by jones & bartlett.
 Lee, W. H. et al. Human retinoblastoma
susceptibility gene: cloning, identification,
and sequence. Science 235, 1394–1399
(1987).
 https://en.wikipedia.org/wiki/Cell_cycle