in detail presentation for anyone who needs to understand cell cycle in detail

1. CELL CYCLE
Topic Review By
Dr.Chitresh Aggarwal
Department Of Medical Oncology

2.  Introduction
 Historic events of cell cycle discovery
 Phases of cell cycle
 Cell cycle machinery
 Steps of cell cycle and its regulation in normal
cells
 Check points of cell cycle
 Cell cycle in malignant cells
 Applied aspects of cell cycle in oncology
 Summary

3. • Definition - The cell cycle is the series of events that take place in
a cell leading to its division and duplication (replication) that
produces two daughter cells
• Specific populations retain the ability to proliferate throughout the
adult life span, which is essential for proper tissue homeostasis.
• Quiescent cell — biochemically and functionally active but do not
divide to generate daughter cells.
• Most cells in the adult body are quiescent
• On average, about 2 trillion cell divisions occur in an adult human
every 24 hours (about 25 million per second).

4. Historic events of cell cycle
discovery

5. Rudolf Carl Virchow
(1855)
“Omnis cellula e cellulae”
(cells only arise from pre- existing
cells)
rejection of the concept of
spontaneous generation by cells

6. Leland Hartwell Paul M. Nurse Timothy Hunt
concept of
“checkpoints”
1970
cyclin-dependent
kinase (CDK)
1976
cyclins
1982

7. The Nobel Prize in Physiology or Medicine 2001 was
awarded jointly to Leland H. Hartwell, Tim Hunt and Sir Paul
M. Nurse "for their discoveries of key regulators of the cell
cycle

8. Overview of the Cell Cycle

9. • Cell moves from the quiescent (also known as G0) state into the first
gap phase, or G1, in which the cell prepares itself for the cell division
process.
• Not surprisingly, this process therefor takes a significant amount of
time (from 8 to 30 hours) and energy.
• Mitogenic growth factors are essential for continued passage
through the G1 phase.
• If growth factors are withdrawn at any point during this phase, the
cell will not divide.

10. • As the cell nears the end of the G1 phase, the cell passes through a
key transition point, called the restriction point, whereupon it
becomes growth factor independent and is fully committed to
undergoing cell division
• Within an hour or two, the cell enters the synthesis phase, or S
phase, in which each of the chromosomes is replicated
• The cell then enters a second gap phase, called G2, which lasts 3 to 5
hours, and then initiates mitosis, or the M phase, a rapid phase
(lasting about 1 hour) in which the chromosomes are segregated.
• Upon completion of mitosis, the daughter cells can enter quiescence
or initiate a second round of cell division, depending on the milieu.

12. CELL CYCLE REGULATION

14. Cyclin-Dependent Kinases (CDK)
and Their Regulators

15. • The Cdks are subfamily of kinases that are defined by their
dependence on a regulatory subunit, called a cyclin.
• Cdks act in association with a cyclin subunit that binds within the
kinase.
• The first identified human Cdk was Cdk1 .
• Cdk4 and Cdk6 regulate cell cycle entry
• Cdk2 may have specific roles during the G1-to-S transition and S
phase.
• Cdk1 is essential in the control of G2 and mitosis and also may play
additional roles in earlier stages.
• Four distinct subclasses—D-, E-, A-, and B-type cyclins—are involved
in cell cycle regulation

18. • There are 3 major mechanisms of cyclin-CDK complex regulation.
• Firstly, The kinase activation is dependent on phosphorylation of a
threonine residue that is adjacent to the active site (Thr160 in Cdk2).
• This phosphorylation is catalysed by a kinase, called Cdk-activating
kinase (CAK)
• In mammalian cells, phosphorylation occurs after cyclin binding.
• Although it appears that at least two mammalian CAKs exist, the
major CAK is a tri molecular complex composed of Cdk7, cyclin H, and
Mat1.

20. • Secondly, the cyclin-Cdk complex frequently is subject to inhibitory
phosphorylation of Thr14 and Tyr15 residues within the Cdk’s active
site by the Wee1 (Tyr15) and Myt1 (Thr14 and Tyr15) kinases.
• Activation of the cyclin/Cdk complex is then dependent on the
action of a dual-specificity phosphatase called Cdc25.
• Mammalian cells have three different Cdc25 proteins Cdc25a,
Cdc25b, and Cdc25c, which show some specificity for different
cyclin-Cdk complexes.

22. • Thirdly, Cdks are modulated by a series of CdK inhibitors (CKIs)
• The CKIs can be divided into two distinct families based on their
biological properties.
• The first CKI family is named INK4, based on their roles as Inhibitors
of CDK4.
• The INK4 family has four members called p16INK4a, p15INK4b,
p18INK4c, and p19INK4d
• These INK4 proteins specifically prevent the binding of cyclins to
monomeric Cdk4 and Cdk6 but do not inhibit other Cdks.
• The second CKI family is named Cip/Kip and includes three members:
p21Cip1 , p27Kip1, and p57Kip2
• Cip/Kip proteins do not bind to monomeric Cdks but associate with
and inhibit the activity of cyclin-Cdk complexes already formed.

25. Retinoblastoma Proteins and
E2F Transcription Factors

26. • The retinoblastoma protein (pRb) behaves as a classic tumor
suppressor
• RB1 gene is inactivated in approximately one third of all sporadic
human tumors.
• pRb and the pRb-related proteins p107 and p130 are collectively
known as the pocket proteins
• These are transcriptional repressors whose major function is to
inhibit the expression of cell-cycle related proteins
• This suppressive activity is dependent on the ability to prevent cell
cycle entry through inhibition of the E2F transcription factors
• The E2F proteins regulate the cell cycle dependent transcription of
core components of the cell cycle control

27. pRb regulates E2F through two distinct mechanisms
. Directly associates with E2F
and it is sufficient to block
the transcriptional activity of
E2F
pRb-E2F complex can recruit
histone deacetylases to the
promoters of E2F-responsive
genes and thereby actively
repress their transcription
• As Cell cycle entry requires the phosphorylation of pRb by cyclin-Cdk
complexes and the consequent dissociation of pRb from E2F,above
two mechanisms do not allow cell cycle entry

28. Retinoblastoma Proteins and E2F Transcription
Factors

30. Cell Cycle Phosphatases

31. • In eukaryotes, two major complexes, PP1 and PP2A, account for more
than 90% of protein phosphatase activity.
• These protein families cooperate in the dephosphorylation
• PP1 and PP2A are major phosphatases responsible for pRb
dephosphorylation during mitotic exit
• Cell cycle ultimately is regulated by the dynamic equilibrium between
Cdks and phosphatases activity.
• In the absence of Cdk activity, the balance tilts in favor of the
phosphatases.
• When Cdks are activated, phosphatase activity is overtaken.
• Reactivation of PP1 and PP2A phosphatases is a mandatory step for
the exit from mitosis and the transition to interphase

32. Ubiquitin-Dependent
Protein Degradation

33. • ubiquitin-mediated protein degradation is a major regulatory
mechanism to ensure ordered transition through the different
phases of the cell division cycle.
• SCF and APC/C are the ubiquitin ligases which drive the degradation
of cell cycle regulators to accomplish irreversible cell cycle
transitions
• Once SCF binds its substrate, it transfers a ubiquitin molecule within
the target protein which targets the substrate to the proteasome for
degradation.
• The APC/C is a much larger complex but has similar mechanism of
action

35. Entry into the Cell Cycle

36. 1
• In quiescent cells , E2F-responsive genes recruit pocket
proteins, along with their associated histone
deacetylases, to actively repress their transcription
2
• CKIs normally are expressed in quiescent cells
• D-type cyclins are present at very low levels in most
quiescent cells
3
• Now when mitogenic signal comes,mitogens directly
induce the transcription of D class cyclins
• Transcriptional induction of D-type cyclins promotes cell
cycle entry.

37. D-type cyclins
associate
with Cdk4
and Cdk6
This complex
phosphorylate
pRb, partially
inactivating its
transcriptional
suppressor
function
the D-
type
cyclins
inactivate
CKIs
Entry into the
cell cycle

39. DNA Replication

40. • The DNA replication machinery is to ensure that the genome is
copied once—and only once—in each cell cycle.
• This is achieved through a two-step process
• first it establishes a pre replication complex (pre-RC) at origin of
replication, a process that is frequently referred to as origin licensing
• Subsequently it transforms pre-RCs into the preinitiation complex
(pre- IC) that activates DNA replication
• These two steps occur at distinct stages of the cell cycle to ensure
that origins are only licensed once per cell cycle and re replication
cannot occur.
• Pre-RC formation takes place during G1.

41. • The first step is the recruitment of the multiprotein complex called
the origin recognition complex (ORC) to the origin DNA
• The origin recognition complex recruits additional proteins including
Cdc6, Cdt1, and finally the mini chromosome maintenance (MCM)
complex, a helicase that is required to unwind the DNA strands to
form the pre-RC.
• Once cells enter S phase, the transformation of the pre-RC to the
pre-IC requires the activity of two kinases: a Cdk and the Ddf4-
dependent kinase
• The transition from pre-RC to pre-IC results in inhibition of Cdt1 by
ubiquitin-mediated degradation and geminin binding.
• Origin licensing cannot occur again until activation of anaphase-
promoting complex/cyclosome (APC/C) at the end of mitosis allows
accumulation of Cdt1.

42. PRE RC COMPLEX
ORC COMPLEX
PRE INITIATION
COMPLEX
ACTS AS A
HELICASE
AND
UNWINDS
DNA

43. Mitosis

46. CELL CYCLE CHECKPOINTS

49. G1/S Checkpoint
• In G1 cells, double-stranded DNA breaks (DSBs) are the most
common and most deleterious type of DNA damage.
• The central components of the DNA damage response (DDR) are two
members of the phosphoinositide 3-kinase-related kinase family
Ataxia telangiectasia mutated (ATM) ATM-rad3-related (ATR)
• The DSBs are recognized by the multifunctional Mre11-Rad50-Nbs1
(MRN) complex.
• MRN complex recruits ATM to the site of damage.
• The active ATM recruits proteins modify the
chromatin at the region of the break activate repair and
signaling Signals Chk2

50. Chk2 influences the G1 cell cycle arrest via two mechanisms
Chk2 phosphorylates all three members of the Cdc25 family
Phosphorylation of these cdc25 family proteins
prevent the activation of cyclin dependent kinases
stop the progression of cell cycle
(rapid response & effect within minutes after DNA damage)
Chk2 phosphorylates p53

51. NORMAL CELL
• p53 protein is maintained at
low steady-state levels because
it has a very short half-life.
• This short half-life is a result of
rapid ubiquitination of p53 by
hdm2 (the human ortholog of
murine mdm2 protein)
degradation of p53.
DIVIDING CELL DURING
G1/S CHECKPOINT AND
Chk2 ACTIVITY
• Phosphorylation of p53 by chk2
prevent association hdm2
• leads to an accumulation of p53
• P53 induces cell cycle arrest
• This p53-mediated arrest takes
longer to develop than does the
cdc25 response
• p53 has the capacity to induce
apoptosis

54. Intra-S Phase Checkpoint
• The major goal to prevent the replication of damaged
DNA
• S phase cells must respond virtually instantaneously to DNA damage
to halt initiation of new replication forks throughout the S phase
• In contrast to DSB in G1 cells , where ATM is solely responsible for
checkpoint activation, in S and G2 checkpoints, recruitment of ATR
also occurs.
Replication-linked DSBs
• S PHASE DNA BREAKS
Non replication linked DSBs

55. Replication - linked DSBs
• the presence of single-stranded
DNA (ssDNA) is a hallmark of
the replication process.
• The ssDNA is coated by
replication protein A (RPA) and
bound by ATR even during the
normal replication process.
• In response to DNA damage,
the ATR kinase is activated, and
it then recruits a variety of
complexes that mediate both
repair and checkpoint
activation, including ATM
Non replication-associated
DSBs
• In first step,these recruit and
activate ATM through the MRN-
dependent process described
previously for the G1/S
checkpoint.
• Then through the action of the
MRN endonuclease it is then
bound by RPA and ATR
• ATR contributes to the
checkpoint response as it
activates Chk1, which also can
phosphorylate the Cdc25
proteins and p53

57. G2 Checkpoint
• G1/S and intra-S phase checkpoints prevent cells from unfaithful
replication
• G2 checkpoint is required to prevent the passage of DNA lesions to
the two daughter cells during mitosis
• DSBs are detected exactly as described previously for the S-phase
non replication associated DSBs.

59. Spindle Assembly Checkpoint (SAC)

60. The kinetochore is the protein structure on chromatids where the spindle
fibers attach during cell division to pull sister chromatids apart.

61. Spindle Assembly Checkpoint (SAC)
• To ensure appropriate partitioning of the chromosomes occurs
during mitosis.
• Chromosome segregation does not occur until all condensed sister
chromatid pairs are aligned at the metaphase plate with the
appropriate bi orientation
• This process actually is controlled by a signaling network that
constitutes the SAC
• The core components of this checkpoint are Mad1, Mad2, BubR1,
Bub1
• During prometaphase, these proteins localize to the outer
kinetochore and, in the absence of biorientation, prevent the Cdc20
activator from binding to the APC/C. (to promote to anaphase)

62. Lack of tension or lack of attachment at the kinetochore
stable Mad1-Mad2 complexes
This is able to bind to Cdc20
The Mad2-Cdc20 association triggers the recruitment of BubR1-Bub3
into an APC/C-inhibitory complex (the mitotic checkpoint complex
[MCC]).
a single unattached kinetochore is sufficient to form these complexes
and inhibit APC/C-Cdc20 activity.
separase is inhibited by the high levels of securin
cyclin B–Cdk1 complexes, being unable to cleave the centromeric
cohesin

63. When all chromosomes are bi-polar attached to the mitotic spindle
SAC is satisfied
Mad1 : Mad2 complex is removed from the kinetochores
Cdc20 is now released from the MCC complex
activates the APC/C
rapid ubiquitination and degradation of cyclin B and securin
Inactivation of these two proteins
activation of separase cleaves cohesion of chromatids

65. CELL CYCLE DEREGULATION IN
HUMAN CANCERS

66. Unscheduled Cell Cycle Entry in Cancer
• Loss or mutation of the prb tumor suppressor – retinoblastoma,
osteosarcoma, small-cell lung cancers
• Overexpression of cyclins - 50% of invasive breast cancers have
elevated cyclin D
• Cdk4 and cdk6 amplification - breast cancers, sarcomas, gliomas, and
melanomas
• CKI - decreased expression of the p57kip2 is found in human bladder
cancers.
• Germline mutations in p16ink4a - predispose to melanoma
• Deletion of p15ink4b and p16ink4a- lymphomas, mesotheliomas

67. Mutations in p53 and Checkpoint Regulators
• The most frequently altered cell cycle
• germline mutations of p53 - Li-Fraumeni syndrome with significantly
increased rates of brain tumors, breast cancers
• Mdm2 gene amplification - resulting in Mdm2 protein
overexpression and subsequent p53 inactivation
• ATM mutations - ataxia-telangiectasia, elevated incidence of
leukemias, lymphomas, and breast cancer
• Chk2 mutations - several cancers, including lung cancer
• Chk1 mutations - human colon and endometrial cancer

68. Aneuploidy and Chromosomal Instability
• Abnormal chromosome number - “aneuploidy” is a frequent feature
of cancer cells
• Estimates suggest that normal cells mis -segregate a chromosome
every hundred cell divisions
• This rate is dramatically increased in cells that display Chromosomal
instability, which missegregate a chromosome in every one to three
cell divisions
• Several regulators such as separase, securin, condensins, Cdc20, or
Aurora kinases, as well as the SAC component Mps1, are included in
the overexpression signature that marks chromosomally unstable
cancers.

70. Cell Cycle In
Oncology

73. Summary

74. • Most cells in postnatal tissues are quiescent. Exceptions include
abundant cells of the hematopoietic system, skin, and gastrointestinal
mucosa, as well as other minor progenitor populations in other
tissues.
• Many quiescent cells can re enter into the cell cycle with the
appropriate stimuli, and the control of this process is essential for
tissue homeostasis
• The key challenges for proliferating cells are to make an accurate copy
of the 3 billion bases of DNA (S phase) and to segregate the
duplicated chromosomes equally into daughter cells (mitosis)
• Progression through the cell cycle is dependent on both intrinsic and
extrinsic factors, such as growth factor or cytokine exposure, cell-to-
cell contact, and basement membrane attachments

75. • The internal cell cycle machinery is controlled largely by oscillating
levels of cyclin proteins and by modulation of cyclin-dependent kinase
(Cdk) activity. One way in which growth factors regulate cell cycle
progression is by affecting the levels of the D-type cyclins, Cdk activity,
and the function of the retinoblastoma protein
• Cell cycle checkpoints are surveillance mechanisms that link the rate
of cell cycle transitions to the timely and accurate completion of prior
dependent events. p53 is a checkpoint protein that induces cell cycle
arrest, senescence, or death in response to cellular stress
• Checkpoints minimize replication and segregation of damaged DNA
or the abnormal segregation of chromosomes to daughter cells, thus
protecting cells against genome instability.
• Disruption of cell cycle controls is a hallmark of all malignant cells.
alterations include dysregulation of the core cell cycle machinery,
and/or disruption of cell cycle checkpoint controls

76. THANK YOU FOR YOUR ATTENTION
CELL