Biomol Lecture 12 Cell Cycle

MOLECULAR BIOLOGY (FAF 2771)
WEEK 12
NOVEMBER 2022
Cell division,
cell cycle, and cancer
Modified by Prof. Edy Meiyanto, Dr. Adam Hermawan, Dr. Muthi Ikawati
Sixth Edition
963
Molecular Biology 3
Bruce Alberts CHAPTER
17
CHAPTER 17:
Alexander Johnson
The Cell Cycle Julian Lewis

David Morgan CELL CYCLE
Martin Raﬀ
Keith Roberts
The only way to make a new cell is to duplicate a cell that already
Peter exists.
WalterThis sim- IN THIS CHAPTER
ple fact, first established in the middle of the nineteenth century, carries with it a
profound message for the continuity of life. All living organisms, from the unicel- OVERVIEW OF THE CELL CYCLE
lular bacterium to the multicellular mammal, are products of repeated rounds of
cell growth and division extending back in time to the beginnings of life on Earth THE CELL-CYCLE CONTROL
over three billion years ago. SYSTEM
A cell reproduces by performing an orderly sequence of events in which it
duplicates its contents and then divides in two. This cycle of duplication and divi- S PHASE
With problems by
sion, known as the cell cycle, is the essential mechanism by which all living things
reproduce. In unicellular species, such as bacteria and yeasts, MITOSIS
Johneach cell division
Wilson
produces a complete new organism. In multicellular species, long and complex
CYTOKINESIS
sequences of cell divisions are required to produce a functioning
Tim organism.
Hunt Even
in the adult body, cell division is usually needed to replace cells that die. In fact, MEIOSIS
each of us must manufacture many millions of cells every second simply to sur-
vive: if all cell division were stopped—by exposure to a very large dose of x-rays, CONTROL OF CELL DIVISION
for example—we would die within a few days. AND CELL GROWTH
The details of the cell cycle vary from organism to organism and at different
times in an organism’s life. Certain characteristics, however, are universal. At a
minimum, the cell must accomplish its most fundamental task: the passing on
of its genetic information to the next generation of cells. To produce two geneti-
cally identical daughter cells, the DNA in each chromosome must first be faith-
fully replicated to produce two complete copies. The replicated chromosomes
2015
must then be accurately distributed (segregated) to the two daughter cells, so that
each receives a copy of the entire genome (Figure 17–1). In addition to duplicat-
Molecular Biology 4
Outline
⬥ Introduction
⬥ Eukaryotic cell cycle
⬥ General regulation of cell cycle
⬥ Regulation of G1/S transition
⬥ Regolation of G2/M phase
⬥ DNA damage induced G1 cell cycle arrest
⬥ Flowcytometry for cell cycle analysis
Molecular Biology 5
Introduction
Molecular Biology 6
Cell cycle and anticancer chemotherapy:

at a glance
https://youtu.be/VRhz3DhjG5M
Signal transduction and cell cycle connection Molecular Biology 7
[Hanahan & Weinberg, 2011]

Molecular Biology 8
Molecular Biology 9
The life of living cells

Three types of proliferative cells:
1. Always dividing
2. Quiescent (Go)
3. Terminally differentiated
Proliferation, establishment, differentiation and
reversal of terminal differentiation are
dependent on serum factors and
oncoproteins
[Stein, et al., Ed. The molecular basis of cell cycle and growth control, Wiley-Liss, Inc. 1999;
Cooper, GM, The Cell, a Molecular Approach, ASM Press, 2001]
Molecular Biology 10
Consideration
⬥ How the cell duplicate its content?
⬥ How the cell divide into two and split it?
⬥ How to regulate those processes?
Outline
⬥ Introduction
⬥ Regulation of G2/M phase
13
Eukaryotic Cell Cycle
18_02_four_phases.jpg
Eukaryotic cell cycle

Four phases
⬥ M phase (Mitotic phase)
⬥ G1 phase (Gap 1)
⬥ S phase (Synthesis phase)
⬥ G2 phase (Gap 2)
15
17
18
Identify the stages!
?
early, middle, & late prophase
? ? ?
late prophase metaphase anaphase
? ? ?
late anaphase telophase telophase & cytokinesis
The stages of mitosis
https://youtu.be/VGV3fv-uZYI
Outline
⬥ Introduction
22
23
https://www.nobelprize.org/prizes/medicine/2001/press-release/
https://www.youtube.com/watch?v=eBsS8X7Wq9E
⬥ The master controllers of events in cell

cycle are a small number of
heterodimeric protein kinases that
contains:
⬩ regulatory subunit (cyclin) and
⬩ catalytic subunit (cyclin-dependent
kinase; CDK)
http://medweb.bham.ac.uk/bmedsci/bms2/chime/structure/2A.gif
Fig 17-16 Cyclin-Cdk complex of the cell-cycle control system
Figure 17-16 Molecular Biology of the Cell (© Garland Science 2008)

Level of cyclins during the cell cycle

Cyclin/cyclin dependent kinases Molecular Biology 32
in cell cycle
Outline
⬥ Introduction
⬥ Regolation of G2/M phase
18_25_growth_factors.jpg
(G1-S check point)

G1-S transition
by Cyc-E/A
Rb pathway
Rb/p53 and HPV (Human Papiloma Virus)

Cell cycle target

chemotherapeutics
Cell cycle target chemotherapeutics: Molecular Biology 41
G-1 inhibitors
[Cells 2019, 8(4), 321;

https://doi.org/10.3390/cells804
0321]
Outline
⬥ Introduction
G1-S check point
and cell cycle arrest
The role of p21 and p27

The role of p53 in the check point

p53
Outline
⬥ Introduction
Mitosis dynamics
49
The role of Molecular Biology 50
CycB-CDK1 complex
in mitosis
50
51
Cellular changes
induced by MPF
MPF:
Maturation Promoting Factor
Cell cycle M-phase regulation Molecular Biology 52
Prometaphase
Degradasi cycB
The role of separase enzyme in chromosome segregation
Cell cycle target chemotherapeutics: Molecular Biology 54
CDK inhibitors
Chemotherapeutics target on mitosis Molecular Biology 55
56
56
Tubulin inhibitor Molecular Biology 57
Outline
⬥ Introduction
⬥ General Regulation of cell cycle
profase akhir DNA arrangement

profase Metafase
Anafase
DNA dibagi menjadi 2 ditarik ke kutub telofase
60
Flowcytometry
Flowcytometer
instrument
Optical design
PMT 5
PMT 4
Sample
PMT 3
Dichroic
Flow cell Filters
PMT 2
Scatter PMT 1
Sensor
Laser
Bandpass
Filters
Dyes for DNA cell cycle analysis

⬥ Propidium iodide
⬦ Excited at 488 nm; fluoresces red (617 nm)
⬦ easily combined with fluorescein stain
⬦ also stains RNA
⬥ DRAQ5
⬦ Max. excitation at 646 nm; can be excited at 488 nm; fluoresces
in deep red at 680 nm max
⬦ Taken up by live cells
Dyes for DNA cell cycle analysis

⬥ Hoechst dyes
⬦ excited by UV; fluoresce blue
⬦ DNA specific - bind to AT
⬦ Hoechst 33342 can be used to stain viable cells
⬥ DAPI
⬦ excited by UV; fluoresce blue
⬦ DNA specific
Definitions & terms

⬥ Ploidy: related to the number of chromosomes in a cell
⬥ Haploid: number of chromosomes in a gamete (germ cell) is called
the HAPLOID number for that particular species
⬥ Diploid: the number of cells in a somatic cell for a particular species
⬥ Hyperdiploid: greater than the normal 2n number of chromosomes
⬥ Hypodiploid: less than the normal 2n number of chromosomes
⬥ DNA tetraploidy: containing double the number of chromosomes
⬥ DNA index: the ratio between the mode of the relative DNA content of
the test cells (in G0/G1phase) to the mode of the relative DNA content
in normal G0/G1 diploid cells
⬥ Coefficient of variation - CV: the ratio between the SD of the mode of
the G0/G1 cell populations expressed as a percentage
Normal 300
cell cycle G0 - G1G0
M G0
G2
225
G1
Cell Count
s
150
75
s G2 M
0
0 200 400 600 800 1000
2N 4N
DNA Content
A typical DNA histogram 2n 4n

G0-G1 G2-M
S
# of Events
Fluorescence Intensity
Flowcytometry of apoptotic cells

Apoptotic cells
Normal G0/G1 cells
# Events
PI - Fluorescence
Cycle cell detection R1
Why cell cycle is important?

Doublets
• Cell replication is controlled by regulating the timing
of nuclear DNA replication and mitosis
Ungated
• The cell-cycle control system regulates the entire
process
• A great analogy is to think of it as a washing machine
control knob
• Cannot implement the next program until the first one Gated
in completed
• Feedback systems operate to provide information on
how processes are progressing
Cycle cell detection Molecular Biology 70
S Phase
DNA/RNA Probes:
• Propidium Iodide
• Ethidium Bromide
• Hoechst dyes
• Acridine Orange G1 G2/M
• Pyronin Y
• Styryl Dyes eg LDS-751
• Mithramycin, Chromomycin
• 7 Aminoactinomycin D (7AAD)
• Diamino-2-phenylindole (DAPI)
• DRAQ5
• Cyanine dyes eg TO-PRO-3,
SYTO/SYTOX dyes Note linear scale! DNA Content
These dyes are stoichiometric –
number of bound molecules are
equivalent to the number of DNA Basic quantitaion of cell cycle phase
molecules present DNA stained with propidium iodide
Cycle cell detection Molecular Biology 71
Example 3: G2/M block Example 2: S phase block

T0 T0
T24 T24
G1 S G2/M G1 S G2/M
O O 72
HO OH
OCH 3 Curcumin OCH3
O O O
H 3CO H 2C CH 2
H
HO
O O
Cancer Epidemiology, Biomarkers & Prevention vaniline
(o-OCH3-p-OH-benzaldehyde) H 2C CH 2
2,4-pentanadione
(acetyl acetone)
O
H3 C CH3
Curcumin undergoes extensive metabolic conjugation & reduction in
HO
G.I tract. OH
CH 3 CH 3
(Cancer Epidemiology, Biomarkers & Prevention, 2002)
PGV-1
H 3C O
H
- Stabile against light and pH HO

O
- Stronger growth inhibitory effect H3 C
- Stronger anti-inflammatory effect HO

H
O
73
PGV-1 1 uM
G1 : 49.12%
S : 33.04%
G2 : 17.84%
Debris: 3.77%
PGV-1 2.5 uM PGV-1 5 uM PGV-1 10 uM

G1 : 45.87% G1 : 39.48% G1 : 45.97%
S : 32.92% S : 31.52% S : 19.99%
G2 : 21.21% G2 : 29.00% G2 : 34.04%
Untreated WiDr cells 24 h Debris: 5.46% Debris: 10.29% Debris: 19.69%
G1 : 50.85%
S : 36.11%
G2 : 13.04%
Debris : 2.54%
74
H 2N
N
NH 2
HN NH
O N H3C HN N
NH O NH
NH N Timidilat N
sintetase NH
O N O + O N O +
dRibosa-5P O NH dRibosa-5P
O O NH
dUMP -
O OH dTMP -
OH
O O
O
O
5,10-metilen THF dihidrofolat

F F
NH Timidin F
Timidin NH
kinase NH
fosforilase
O N O O N O
O N O
H dRibo sa
dRibosa-5P
5-FU 5-FUdR
5-FdUMP
5-FU is S-phase active agent
5-FU main target à Thymidylate synthase activity à S-phase arresting agent 40

75
5-FU 1 mM 5-FU 1 mM+

G1 : 52.89% PGV-1 2.5 uM
S : 45.15% G1 : 32.77%
G2 : 1.96% S : 58.51%
Debris: 3.83% G2 : 8.73%
Debris: 5.55%
5-FU 1 mM+ 5-FU 1 mM+

5-FU 1 mM+ PGV-1 7.5 uM PGV-1 10 uM
PGV-1 5 uM G1 : 13.97% G1 : 11.68%
G1 : 20.86% S : 70.23% S : 72.07%
S : 65.79% G2 : 15.8%
Untreated WiDr cells 24 h G2 : 16.25%
G2 : 13.35% Debris: 10.12% Debris: 12.56%
G1 : 50.85% Debris: 8.48%
S : 36.11%
G2 : 13.04%
Debris : 2.54%
Thank you
edy_meiyanto@ugm.ac.id
muthi_ikawati@ugm.ac.id

Biomol Lecture 12 Cell Cycle

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MOLECULAR BIOLOGY (FAF 2771)

Bruce Alberts CHAPTER

The Cell Cycle Julian Lewis

Cell cycle and anticancer chemotherapy:

[Hanahan & Weinberg, 2011]

The life of living cells

Eukaryotic cell cycle

The stages of mitosis

⬥ The master controllers of events in cell

Fig 17-16 Cyclin-Cdk complex of the cell-cycle control system

Figure 17-16 Molecular Biology of the Cell (© Garland Science 2008)

Level of cyclins during the cell cycle

(G1-S check point)

Rb/p53 and HPV (Human Papiloma Virus)

Cell cycle target

[Cells 2019, 8(4), 321;

The role of p21 and p27

The role of p53 in the check point

profase akhir DNA arrangement

Dyes for DNA cell cycle analysis

Dyes for DNA cell cycle analysis

Definitions & terms

A typical DNA histogram 2n 4n

Flowcytometry of apoptotic cells

Normal G0/G1 cells

Cycle cell detection R1

Why cell cycle is important?

Example 3: G2/M block Example 2: S phase block

- Stabile against light and pH HO

- Stronger growth inhibitory effect H3 C

- Stronger anti-inflammatory effect HO

PGV-1 2.5 uM PGV-1 5 uM PGV-1 10 uM

5,10-metilen THF dihidrofolat

5-FU is S-phase active agent

5-FU main target à Thymidylate synthase activity à S-phase arresting agent 40

5-FU 1 mM 5-FU 1 mM+

5-FU 1 mM+ 5-FU 1 mM+

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