Cell Cycle and Cell Death Mechanisms OS 201

Paul Mark B. Medina, PhD Exam 6

06 Nov 2019 Trans 1

OUTLINE  Those with mixoploidy are viable unlike those with

I. Cell Cycle III. Cell Death Mechanisms polyploidy
A. Main Purpose of Cell A. Cell Death  Mixture of cells that are mostly 2n and some 3n
Cycle B. Apoptotic Markers  Die early on in development
B. Somatic Cell Division C. The Apoptotic Pathways  Aneuploidy: Addition or subtraction of one or more single
C. Gametic Cell Division D. Control of Cell Numbers chromosomes (e.g. 2n+1, 2n-1, 2n+2, etc.)
II. Cell Cycle Regulation and Cell Size  Observed in genetic syndromes involving sex chromosomes,
A. Cell Cycle Length E. Deregulation of such as Klinefelter’s syndrome (2n+1), Turner’s syndrome
B. Cell Cycle Checkpoints Apoptosis in Malignancies (XO, 2n-1), Down syndrome (2n+1)
C. Signal Molecules in F. Other Cell Death  The X chromosome is a very important chromosome for
Cytoplasm Regulate Cell Mechanisms survival. (No X chromosome = lethal
Cycle IV. References  Monosomic aneuploidy usually persists, whereas trisomic
D. Proto-Oncogenes and V. Appendix aneuploidy are more often lethal. Human cell can tolerate
Tumor-Suppressor Genes mostly monosomic cells than trisomic.
Note: This trans is based from Dr. Medina’s lecture, power point
presentation, and 2023 trans. B. SOMATIC CELL DIVISION
 Interphase - 95% of cell cycle
I. CELL CYCLE  Involves everything but the M phase
 “A cell in a bicycle” – a cycle of different phases divided into 2
 Organelle duplication, DNA replication, growth
major divisions:
 3 phases: G1, S, G2; no division occurs
 M phase – further divided into two:
 Mitosis  Go phase
 Cytokinesis  Long term transient state depending on microenvironment
and if conditions are ideal
 Interphase
 Cell is quiescent and may exit here
 Duration of cell division varies based on cell type
 Cell cycle arrest
 Typically takes 24 hours in humans but can vary from 8  Haven’t gone through S phase therefore no duplication
hours to over a year  Happens before the restriction point – committing step
 Primary determinant is G1 phase (most variable phase in of the cell cycle
terms of time)  M Phase – 5% of the cell cycle
 Mammalian cells:  Consists of Mitosis + Cytokinesis
 G1 (8-10 hours)  These two stages occur separately and must not be taken as
 G2 (4-6 hours) one
 S (6-10 hours)
 Mitosis
 M phase (minutes only)
 Nuclear division resulting in two nuclei identical to each
other and the parental nuclei as well
A. MAIN PURPOSE OF CELL CYCLE  Not all mitosis that do occur result in cytokinesis
 To accurately transmit genetic information  Some of your cells are multinucleated (e.g. Syncytium of
 To maintain normal ploidy (i.e. diploidy in humans) skeletal muscles)
 Ploidy: number of set of chromosomes in a cell, or in the  Cytokinesis
cells of an organism  Cytoplasmic division resulting in two daughter cells
 Strawberry: 8n (n=7, 56 chromosomes)
 Apple: 2n, 3n, 4n (n=17, 34/51/68 chromosomes)
 Oyster: 2n, 3n (n=10, 20/30 chromosomes)
 Human: somatic cells are diploid (2n = 46 chromosomes),
reproductive cells (sperm and egg) are the only ones which
are haploid (n= 23)

Variations to Ploidy
 Euploidy: Addition of whole chromosome sets (e.g. n, 2n, 3n,
4n = haploid, diploid, triploid, tetraploid, respectively)
→ Polyspermy: Two sperms fertilizing a single egg resulting in
triploidy (3n)
 Results in polyploidy (specifically triploidy) where there is
presence of more than 2 homologous sets of
chromosomes in the organism(Nature.com)
 Occurs 2-3% of the time in human pregnancies
 Causes 15% of miscarriages
 No fully developed individual as a product of polyspermy
 Some may come to term but die immediately upon birth
→ Mixoploidy: Presence of cell lineages with different ploidy
states coexisting in an individual(Destouni, et al., 2016)

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Figure 1. Cell cycle overview
Interphase
1. G1 Phase (Gap 1)
 Organelle duplication but no DNA replication
 Cells are metabolically active but look benign microscopically
 Comes right after mitosis, therefore cellular content is reduced
and must grow in volume
 Duration is variable
→ G1 phase may be short or non-existent in rapidly dividing cells
(embryonic cells and cancer cells)
 Preparatory stage for S phase (i.e. enzymes for DNA replication
are produced in this phase)
 Cells that remain in G1 for a long time go into G0 (permanent
tissues, such as neural tissue)
→ May look the same but are metabolically different
→ G0 leaves the cell cycle in the early part of G1
→ Once a cell exits the cell cycle and enter G0, it may still re-
enter the cell cycle again at G1 even after a long time
Upon external or internal stimuli (e.g. induced pluripotent stem cells)
Non-dividing highly differentiated cells are in G o
 Most variable phase of the cell cycle and therefore determines
the length of cell cycle
 Distribution of organelles happens here, specifically
mitochondria because mitochondria cannot be made from
scratch. It only comes from pre-existing mitochondria.
 Restriction Point – if cell passes this stage, it is committed to
finish the cell cycle by continuing to S phase.
2. S Phase (Synthesis)
 DNA and centrosome (microtubule-organizing center)
replication
 Stage where cell is already committed to cell division and is
irreversible
 Irreversibility actually comes at the later parts of G1, before
going into the S phase(2023)
 Formation of two sister chromatids bound together by the
kinetochore – 1 chromosome: 2 sister chromatids
 The term “chromatid” only applies in the presence of the
other
 DNA replication is semi- conservative: Each new DNA molecule
is composed of one conserved strand from the original template
and one newly synthesized strand(BioNinja)
Figure 2. Semi- conservative DNA replication model
→ Two identical daughter genomes produced
→ Preserves integrity of genetic material
→ Accurate transmission of DNA from mother cell to 2 daughter
cells is of important concern in cell division
→ DNA replication is bidirectional: replication occurs in two
opposite directions, towards each end of the DNA (Alberts 2015, 2023)
 A replication bubble is formed at the origin of the separation of
parental DNA strands(Alberts 2015, 2023)
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 Two replication forks are produced, each moving towards
opposite ends of the DNA (bidirectionally) (Alberts 2015, 2023)
 One strand is replicated from 5'  3' (leading strand), the
other is replicated from 3'  5' (lagging strand)
 Replication and transcription always proceeds in 5’  3’
direction and the template used is being read in the 3’  5’
direction
 Synthesis of polypeptide (translation) occurs from amino end to
carboxyl end
Figure 3. Bidirectional DNA replication where both strands are
simultaneously replicated in two directions.
3. G2 Phase (Gap 2)
 Cell continues to grow (second phase of growth)
 Determining cell stage
 Cells at different stages of the cell cycle can also be
distinguished by their DNA content
 Mitotic spindle begins to form
 Cellular content further increases
 Preparation for M Phase
 Phase where the stage at which the cell is in can be determined
 DNA replication has already occurred  twice the amount of
DNA content compared to G1
 92 chromatids present and 46 chromosomes
 Polyploidy: 4n because there are 4 sets of genomes
M Phase
 Divided into 2 subphases
 Nuclear division (not always followed by cytokinesis and can
result to multinucleated cells)
 Cytoplasmic division
 Together results to cellular division
 Mitosis: nuclear division resulting to 2 nuclei identical to each
other and to the parental nuclei
 Process by which eukaryotic cells (except germ cells
undergoing meiosis) separate the chromosomes in its cell
nucleus
 Division of cellular nucleus is referred to as karyokinesis
1. Prophase
 Chromosomes begin to condense and become visible by light
microscopy
 Two sister chromatids appear to be attached at the centromere
 Two centrosomes (which were duplicated during S phase) begin
to move to opposite ends of the cell
 The centrosomes begin assembly of the mitotic spindle
 Microtubule-containing structure with dense asters forming at
each centrosome
 The microtubules in the cytoskeleton disassociate into tubulin
which is added to the mitotic spindle
 In animal cells (but not plant cells), centrioles are located at the
core of the centrosomes
 Nuclear envelope replication by remains intact
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Figure 6. Metaphase Stage

Figure 4. Prophase of Mitosis 4. Anaphase

 Sister chromatids separate and begin to move to opposite poles
2. Prometaphase  Anaphase A: the chromosomes are pulled by the centromeres
 Not a main phase but is included in some literature (Sir does not towards the poles as the kinetochore microtubules shorten
consider this a major step)  Anaphase B: the poles push apart as the polar microtubules get
 Added due to the many processes occurring in prophase longer
 Nuclear membrane breaks down  Anaphase A and B may occur in this order or at once,
 Centrosomes stop at locations at opposite poles of the cell depending upon cell type
(spindle pole)  Both processes contribute to chromosome segregation
 As the nuclear membrane is no longer present, mitotic spindle
microtubules make contact with the chromosomes via their
kinetochores
 Each chromosome develops two kinetochores, one for each
sister chromatid
 DNA sequence + kinetochore forms the centromere
 The chromosomes are forced toward the center of the cell
 Mitotic spindle microtubules can attach to various microtubules
of the cell
 Kinetochore microtubules: attached to kinetochore
 Polar microtubules: attached to microtubules from the other
pole
 Aster microtubules: attached to proteins of the inner plasma
membrane, helping to position the spindle
Figure 7. Anaphase Stage

5. Telophase
 The daughter chromosomes arrive at the pole and begin to
decondense to chromatin
 The nucleoli develop, the spindle disassembles and the nuclear
envelope reappears forming two nuclei
 Marks the end of mitosis
 Cytokinesis begins with the contraction of the contractile ring
 Before cytokinesis, no chromatids are observed, but only
chromosomes since they are no longer attached to each other

Figure 5. Prometaphase Stage

3. Metaphase
 The chromosomes are completely condensed
 The chromosomes are paused and aligned at the metaphase
plate, a plane half-way between the poles
 Kinetochore microtubules attach sister chromatids to opposite
poles of the spindle
 A mitotic karyotype can be constructed from cells arrested at
metaphase

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Figure 8. Telophase Stage

Required for Transmission of Chromosomes
(M Phase)  High energy dependence
 Cytokinesis is dependent upon the tightening of a contractile
 One and only ONE centromere
ring of actin microfilaments through the action of myosin (and
 To prevent splitting at multiple sites during anaphase ATP) to pinch the cell into two
 Dicentric chromosomes (having two  High amounts of mitochondria can be found around
centers/centromeres) can be pulled apart resulting in a contractile ring to provide readily available ATP for this
broken chromosome process
 Functional telomere at both ends  Cytoplasmic division is not always equal (asymmetric cell
 To prevent ends from sticking to other chromosomes division)  smaller cytoplasm for one daughter cell
 These prevent nuclease degradation  Occurs in normal hematopoietic precursor cells
 Chromosomes must be fully replicated  Mutations lead to asymmetric cell division in malignant
 No missing genes hematopoietic precursor cells
 Chromosomes cannot be too large or too small  If cytokinesis fails or if cell doesn’t undergo cytokinesis →
 Can cause missing or extra genes or other mutations binucleated or multinucleated cells
VIDEO (preferably watched before proceeding):  In human cells: osteoclast, hepatocytes, striated muscles,
https://www.youtube.com/watch?v=DpeRQyNIvak multinucleated giant cells (MGC) in urinary bladder
 Occurs as a normal condition in the body
6. Cytokinesis (Cytoplasm Division)
 Division of the cytoplasm and organelles after the chromosomes
have been sorted to complete the process of cell division
 Not a phase of mitosis (Mitosis is nuclear division; cytokinesis is
cytoplasmic division, usually begins in early anaphase.)
 Cytoplasm separate
 Usually begins in early anaphase
 Animal cells: cytoplasm pinches in forming a cleavage furrow –
takes place in the contractile ring
 Plant cells: cytoplasmic division occurs by the formation of a
cell plate and a new cell wall is formed
 Prokaryotes (bacteria & blue green algae): cytoplasmic division
is by binary fission (no nucleus)
Figure 10. Formation of Cleavage Furrow

C. GAMETIC CELL DIVISION

Note from TG: Not discussed during the lecture so this part of the trans is
adapted from 2023.

Meiosis I

Figure 9. Cytokinesis

 Intermediate filaments (actin, septin, myosin) are attached to

the plasma membrane via anillin
 Actin, septin, PIP2, myosin are molecules involved in
cytokinesis
 Actin: main cytoskeletal molecule involved in the inward
pinching, binds to microtubules
 Phosphatidylinositol bisphosphate (PIP2): high Figure 11. Meiosis I (see Appendix for bigger version of image)
concentration in the area where the contractile ring will form
 Septin cytoskeleton: required for constriction site formation 1. Prophase I
 Anillin serves as a docking station linking these molecules to  Duplicated homologous chromosomes condense
each other (actin, septin, myosin) to concentrate around  Homologous chromosomes pair up at the chiasma and
cleavage furrow exchange segments (chiasmata)
 Pleckstrin Homology domain of anillin associates with  Centrosome movement, spindle formation, and nuclear
septin and membrane phospholipid PI(4,5)P2 which is highly envelope breakdown occur as in mitosis
concentrated at the cleavage area

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2. Metaphase I
 Paired homologous chromosomes line up along the equator of II. CELL CYCLE REGULATION
the cell
 One homologue of each pair faces each pole of the cell and
attaches to spindle microtubules via its kinetochore

3. Anaphase I
 Homologues separate, one member of each pair going to each
pole of the cell
 Sister chromatids do not separate

4. Telophase I
 Spindle microtubules disappear
 Two clusters of chromosomes have formed, each containing
one member of each pair of homologues
 The daughter nuclei are therefore haploid
 Cytokinesis commonly occurs at this stage
 There is little or no interphase between meiosis I and meiosis II
Figure 13. Various checkpoints (the stop and go signals) of each cell
cycle phase
Meiosis II
 Occurs in a similar process as mitosis
A. CELL CYCLE LENGTHS
 Results in 4 haploid cells, each containing one member of each
 Vary by cell type: embryonic cells, stem cells (e.g., blood cells &
pair of homologous chromosomes
epithelial cells), sperm cells
 G1 determines the length of the cell cycle
 S and G2 are about the same length for different cells, before
it goes into the M phase
 G1 is prolonged in stable or permanent cells (exist as G ) 0

 G1 is rapid or non-existent in rapidly dividing cells

→ Example: embryonic cells
 Cell growth is not part of cell cycle in embryonic cell
cleavage
 Number of cells increase without cytoplasmic growth
 All energy goes into DNA synthesis, so G1 (growth stage)
is lacking and G2 is quite short
 Each round of division subdivides original cytoplasm into
smaller and smaller cells, until adult cell size is reached
 Cells are not equal to the size of the mother cell due to
lack/rapidity of G1

B. CELL CYCLE CHECKPOINTS

Figure 12. Meiosis II, separation of sister chromatid. (see Appendix for  Control points where stop and go signals regulate the process
bigger version of image)
G1 Checkpoint
Table 1. Differences between mitosis and meiosis. Restriction (R) point
MITOSIS MEIOSIS
 Point where the cell commits to the cell cycle, continuing to the
Occurs in somatic cells Occurs in germ cells S phase
Nucleus divides only once Nucleus divides twice  Prolonged in stable or permanent cells (called G0)
Four daughter cells are  Growth factor dependent
2 daughter cells are formed
formed  Period during which cells are responsive to mitogenic growth
Daughter cells are diploid Daughter cells are haploid factors (GFs) and transforming growth factor-beta (TGF-β)
Occurs more frequently Occurs less frequently  Mitogenic GFs: Allow the cell to progress through cell cycle
Daughter cells form somatic Daughter cells form  TGF-β: Prevents the cell from progressing through the cell
organs gametes cycle
There is only one prophase, one There are two of each  The cell decides whether to proceed to Go or S phase
metaphase, one anaphase, one phase and 5 subphases in depending on which of the trophic factors it is sensitive to
telophase prophase- 1  If cell is sensitive to TGF-β → do not get past R point
Number of chromosomes are Number of chromosomes
not changed in daughter cells are reduced to fact
G1/S Checkpoint
 Decides if after division, cell will be quiescent or proceed to cell
cycle
 Makes decision of whether the cell should divide, delay division,
or enter a resting stage
 Nutrition dependent

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 Checks the existence of all conditions including nutrients and
enzymes that are required for DNA synthesis
S Checkpoint/DNA Damage Checkpoint
 Cell progression to G2 is halted in presence of gross damage to
genome
 During replication, there can be errors  the cells will halt at the
S phase
G2 Checkpoint
 Located at the boundary between G2 and M phase
 Rapid or non-existent in rapidly-dividing cells
 Proper completion of DNA synthesis is required before cell can
initiate mitosis
 Entrance into M phase is blocked if DNA replication is not
completed
 Point of cell division is accurate genome transmission
 Cell remains in G2 will as DNA repair occurs
M Checkpoint/Spindle Assembly Checkpoint
 Boundary between metaphase and anaphase
 All chromosomes must be properly attached to mitotic spindle
via TF4/kinetochore
 If not properly attached  unequal distribution of
chromosomes between 2 daughter cells
 Again, the goal is accurate transmission of DNA →
everything leads to making sure you have proper distribution
of genome
 Consequences are dire
 Dysfunctional cells which end up dying
 If death mechanism does not ensue → cancer
 Errors result in anaphase block/stopping of mitosis
Figure 14. DNA/Genome- centric checkpoints for cell cycle
progression. Misrepresented figure of what happens, many processes in
the S phase already occurs in G 1, lots of overlap in the cycle and hard to
depict in drawing. Latter part of G1 is the R point.
VIDEO (preferably watched before proceeding): https://tinyurl.com/ybtnb5vp
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C. SIGNAL MOLECULES IN CYTOPLASM REGULATE
CELL CYCLE
Cyclin-Dependent Kinase (CDK)
 CDK exert their effects by phosphorylating specific serine
and/or threonine residues on their protein substrates that
perform the various cell cycle events
 Phosphorylation of protein substrates by CDK can result in
conformational changes that alter their catalytic activity and their
interaction with other proteins
1. Kinase
 Phosphorylates proteins by transferring phosphate group from
high energy molecule (ATP) to amino acid residue of protein
 Named after their substrates
 2 major types:
 Ser/Thr kinase
 adds phosphate to serine or threonine
 Tyr kinase
 adds phosphate to tyrosine
 Both have hydroxyl groups where phosphate can be added
 Phosphate group comes from ATP
 ATP is also a main currency of energy to the cell, a minor
one is GTP
 GTP is the source of energy for translation
 How many GTPs are required to synthesize an amino
acid peptide? 2 GTPs per amino acid.
 Used by cells to regulate cell cycle
 These kinases are inactive unless bound to cyclin molecule
(cyclin dependent)
 CDK (inactive on its own) + Cyclin → active
 CDK- Cyclin complex is equivalent to MPF (maturation
promoting factor)
 Cyclin concentrations rise in G2 and fall during mitosis
 Cyclins begin accumulating late in S phase
 Cyclins are degraded during mitosis
 Cycles in concentration from almost 0 to very high
concentrations between M phase and interphase
 Up just before mitosis, goes down after M
 Goes up during interphase and leads to M after reaching a
certain increase in concentration
 MPF activity peaks before each cell division
 Dependent on cycling amount of cyclin
 Moreover, MPF has kinase activity
Nice to Know!
Cyclin was discovered in sea urchin embryos. Purification of MPF
resulted in the birth of Cyclin Dependent Kinases.
2. Cyclins
 In the first discovery of cyclin, it was called Maturation
Promoting Factor
 Different or “fluctuating” expression throughout cell cycle
 i.e. rise in G2 phase and fall during mitosis
 Note: Be familiar with the following:
 Cyclin D
 Comprises 3 related proteins (D1, D2, D3)
 Binds CDK4 and CDK6
 Cyclin D1 binds CDK’s early to mid G1, and together with
Cyclin E/CDK2 inactivate pRB (cell cycle inhibitor)
 Cyclin E
 Binds to CDK2
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 Cyclin E/CDK2 phosphorylates p27 (Cyclin D inhibitor) which

tags it for degradation; functions in G1 to S transition
 Promotes expression of Cyclin A

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 Cyclin A
 S-phase cyclin
 Binds CDK2 CDK regulation by phosphorylation
 Functions in G1/S transition
 CDK is active when phosphorylated
 Key regulator of cdc25 and CDK1
 Phosphorylation of Y15 comes before T161
 Involved in activating Cyclin B/CDK1 complex
 Conformational changes associated with CDK phosphorylation
 Cyclin B
 Upon binding of the CDK to its corresponding cyclin molecule,
 First to be discovered (where the MPF term came from) the CDK undergoes a conformational change that renders the
 Mitotic cyclin active site more accessible to its substrate
 Binds CDK1 to make MPF (Functions in entry to M phase)  Free CDK: t-loop blocks substrate access
 CDK + Cyclin: binding of cyclin moves t-loop in order to
activate it
 T161 (Threonine 161) phosphorylation: phosphorylation
moves t-loop more, active site more available to substrate
that requires phosphorylation

Figure 15. Cyclin Expression Cycle. The graph illustrates how the
concentration of cyclins generally starts to increase at G1 (or S phase for
cyclin B), but never at G2 phase.

CDK Regulation
Cyclin synthesis and destruction
 In the absence of cyclin, CDK is non- functional Figure 17. G1-S Transition in S. cerevisiae. Cln1/Cdc28 and
 Drives the early embryonic cell cycle Cln2/CDC28 will inhibit SIC1, consequently disabling the inhibition of
 Cyclin destruction is controlled by ubiquitination Clb5/Cdc28, allowing G1-S transition to take place. So, Cln1/Cdc28
(promoter of cell cycle progression) inhibition will inhibit G1-S transition (from
 Particular section of cyclin protein involved in its destruction 2023, not discussed in the 2024 lecture)
.
 Mitotic cyclin destruction box
 Polyubiquitination is another way → targeted for destruction
through a supramolecular structure called proteasome which
cuts them into smaller pieces (not specific to cyclin only)

Figure 16. CDK Regulation by polyubiquitination. The figure (a) shows

the ubiquitination target sites for different cyclins and the sequence within
each. The lower figure (b) shows the general flow of how the CDK is
degraded: ubiquitin “tags” the cyclin which renders it recognizable to
proteasomes which are the effectors of the degradation.
Figure 18. Regulation of CDK by phosphorylation and proteolysis.

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Cyclin Dependent Kinase Inhibitors (CDKI)

 p21 family (p21, p27, p57) inhibits by binding to the active site
of CDK- cyclin complex
 INK4 family (p16, p15, p18, p19) inhibits by replacing cyclin
 Disassembles the complex
 Regulates the G1-S transition
 p16 is the most frequently mutated in human tumors
 Mutation of inhibitor CDK-Cyclin complex by replacing cyclin
allowing cells to continually divide
 Dissociation of CDK- cyclin complex
 Experimental systems important for cell cycle studies
 One and only one replication is controlled by CDKs
 Regulate G1-S transition
 Similarity: yeast and mammals (of G1 control network)
 A lot of what we know comes from yeast, which are very
similar to mammals
 In terms of molecular and physiological functions with
regards to cell cycle
 Regulated by the type of CDK cyclin complex that abounds in
that phase
 Complex forms in different stages which drives movement
from one stage to the next stage and can be inhibited by
cyclin inhibitors

Table 2. Types of cyclins found in vertebrates and yeast.

VERTEBRATE YEAST CYCLINS
CYCLIN-CDK
CYCLINS
G1 CyclinCdk Cyclin D Cln3
G1/S CyclinCdk Cyclin E Cln1, 2
S CyclinCdk Cyclin A Clb5, 6
Figure 20. CDK- Cyclin Complex activity throughout the cell cycle. The
M CyclinCdk Cyclin B Clb1, 2, 3, 4 figure (top) shows the different phases of the cell cycle and the
corresponding CDK-cyclin complexes which are predominating in each
phase, including the inhibitors that act on it. The bottom figure shows the
complexes and the breadth of their predominance across the phases of cell
cycle.

D. PROTO-ONCOGENES AND TUMOR-SUPPRESSOR

GENES
Proto-oncogenes
 Proto-oncogenes - genes that promote normal cell division;
a
regulate cell growth
 Normal cellular function which may not be necessarily related to
the cell cycle
 But if they become mutant forms, they are converted to
oncogenes
 As oncogenes, they gain a new function (gain of function
b
mutation) to drive the cell cycle to keep continuing, promoting
Figure 19. CDK Inhibitors. CKIs can be classified into two families. Both uncontrollable cell division despite presence of check points
act on the CDK-cyclin complex to inactivate the complex but they differ on  Tightly regulated by many factors
how they inactivate. p21 family (a) binds to the complex and plainly
inactivates the complex while the INK4 family (b) separates the complex,  Active = promotes cell cycle
making it inactive.  Inactive = shuts off cell cycle
 Mutated proto-oncogenes

Oncogenes
 Oncogenes - mutant forms of proto-oncogenes; always active
and promotes cancer
 Cell cycle promoter, like the green traffic light
 Due to inactive intracellular signaling protein
 Accelerate cell growth and cell division in cancer cells
 We have two copies of a gene in a cell
 Male version and female version, if either one gets mutated,
that is enough for the cell to rapidly divide uncontrollably
 Normal oncogene may also exist but is overridden by the
mutant oncogene (dominant, other one becomes “recessive”)

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Tumor suppressor genes

 Tumor suppressor genes - act like a brake pedal; prevents The Hallmark of Cancer
cell from dividing when there are damages, function in check  Incidences by which cancer can be promoted
points  Self- sufficiency in growth signals
 Cell cycle stoppers, like the red traffic light.  Evading apoptosis and immune surveillance
 In normal cell: remove/inactivate tumor; prevent cancer  Insensitivity to anti-growth signals
 In cancer cell: mutated/inactivated tumor suppressor gene;  Sustained angiogenesis
damage to genes lead to cancer
 Tissue invasion and metastasis
 Asserts the accuracy of genetic material
 DNA damage stress → limitless replicative potential
 When these are mutated, they cannot stop the cell from moving
through cycle in the presence of errors (Loss of function  Oxidative stress
mutation)  Proteotoxic stress
 Two hit hypothesis - if first hit is a germ line mutation, second  Metabolic stress
mutation more likely to enable cancer
 Both copies of the gene are needed to be mutated and non-
functional in order to lose control of the cell cycle
 Heterozygous condition: Normal function dominant over loss
of function, normal gene balances the mutated gene
 p53 tumor suppressor protein
 Triggers cell suicide
 If cell cannot repair damage → suicide, else it will propagate
and give it to daughter cell → potentially have cancer
 Loss of heterozygosity (heterozygous condition: normal gene
balances the mutated gene)
 P16 is frequently mutated in human tumors
 Regulator of the CDK cyclin complex

Figure 22. Hallmarks of Cancer. The figure shows the various factors or
phenomena which are associated to an increased risk of cancer incidence.
Not much emphasis was given for this but it is helpful to observe how these
are related to each other.

Figure 21. Tumor Suppressor protein blocks signal to cell,

preventing division.

Alvocidib
 Alvocidib (aka Flavopiridol) - flavanoid alkaloid CDK9 kinase
inhibitor under clinical development for the treatment of acute
myeloid leukemia

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Not discussed but is included in 2023 Trans:  Two major forms of cellular death:
Quiescence and Senescence  Necrosis (cellular suicide)
 Apoptosis (programmed cell death
1. Factors that determine the kind of cellular death:
 Nature of the pathogen
 Pathogen load
 Site of infection

Forms of Cellular Death

 Pyroptosis and Necrosis
 Apoptosis and Autophagy

Pyroptosis and Necrosis

Figure 23. mTOR signaling involved in Quiescence and
Senescence
 Quiescence
→ Complete activation of p53 (tumor-suppressor protein)
→ Results from lack of nutrition
→ Inactivates mTOR thereby stopping growth
 Senescence
→ Partial activation of p53
→ Results from stress stimuli and DNA damage
→ Degenerative process ensuing cell death contrary to
quiescence
→ Does not completely inactivate mTOR so cells can still
grow but without division due to signals such as p21 and
p52 (inhibitors of cell division)
 p21: prevents substrate from binding
 p16: disassemble CDK4-cyclin complex
 Pro-inflammatory
 Elicit immune responses (immunogenic)
 Two ways of causing inflammation
 By production and release of cytokines, which are
inflammatory agents
 By membrane disruption releasing DAMPS (Damaged
Associated Molecular Proteins
 DAMPS: released by membrane disruption and are recognized
by the immune system, therefore initiating inflammatory
response
 Pyroptosis requires caspase 1

III. CELL DEATH MECHANISMS

 Zombie genes
Apoptosis and Autophagy
 According to sir, “death is not the end” because certain  Non-inflammatory
genes are still active even after death of the organism →  Apoptosis form vesicular structures called apoptotic bodies
zombie genes which contain organelles or structures
 Active gene expression which happens in animal models and  Apoptotic bodies are engulfed by macrophages
probably humans too  Similarly, autophagy involves engulfing pathogen into a
 Genes involved in inflammation and activation of immune autophagosome
system (necrosis leads to a lot of inflammatory action)  Keep cell membrane intact
 Counteractive stress genes (living cell in a dead host will  Apoptosis requires other caspases except caspase 1
undergo stress)
 Genes that promote cancer (attempt to immortalize the Nice to know!
cells)  DNA fragmentation occurs in apoptosis and pyroptosis,
 Myocardium, pericardial fluid and blood from 30 cadavers in not in oncosis and autophagy
relation to post-mortem interval showed a few genes active
for 12 hours (Villanueva, 2013)
 Discovered by Peter Noble of University of Washington on Similarities and Differences in Cell Death
June 22, 2016
 In mice, 515 genes were seen kicking into gear (being
activated) and were functioning at full capacity up to 24
hours after death
 In zebrafish, 548 genes retained their function for 4 whole
days after the animals had died before showing any signs
of winding down

A. CELL DEATH
 Maintains a relatively constant number of cells in the body
1. Human adult: 50-70 billion cells die each day
2. Average child (8 – 14 y/o): 20-30 billion cells die a day
 Ensures that some cells in the body are removed when
appropriate
→ Humans: each hour we lose billions of cells via apoptosis
→ Most of these are healthy cells which have no defects
 Why? For development and regulation (e.g. B and T cells that do
not pass certain tests are removed)
 Most embryo development involves programmed cell death
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Figure 24. Cell death through Necrosis (Left) and Apoptosis (Right).  Reception of negative signals
 Increased levels of oxidants within cell
Table 3. Comparison of features in apoptosis and necrosis.  Damage to DNA by oxidants
APOPTOSIS NECROSIS  Death activators: Tumor necrosis factor alpha (TNF-a),
Cell shrinkage Cellular swelling Lymphotoxin (TNF-B), Fas ligand (FasL)
Chromatin condensation
Pyknosis, nuclear
(characteristic ladder B. APOPTOTIC MARKERS
disintegration
appearance)  A number of activities take place
Cytoplasmic blebbing, apoptotic Dissolution of cell  Occupation/binding of death receptors
bodies membranes  Dimerization of Bcl-2 family members
No inflammation Inflammation  Release of cytochrome c from mitochondria
Involves whole organs or  Activation of caspases
Single or small groups of cells
large part of organ  Activation of DNAse
 Translocation of phosphatidylserine
Cell Death by Injury (Necrosis)
 Normally oriented towards cytoplasm in bilayer
 Necrosis: accidental cell death
 Flip from inside to outer layer of cell membrane
 Killing of cell, decay or destruction
 ATP-dependency
 Characterized by organelle distension and cell swelling,
 Because apoptosis is programmed as opposed to necrosis
membrane disruption, ATP depletion, and induction of
which does not consume plenty of ATP
inflammation
 Internucleosomal DNA fragmentation (ladder pattern)
 Cell has no plan to die but is killed anyway
 Happens between nucleosome
 Oncosis is sometimes used synonymously with necrosis
 No apoptosis at +4 C and below
o

 Necroptosis: regulated necrosis

 Because apoptosis is an enzyme-dependent process
 May be due to mechanical damage or exposure to toxic
 Low enough will protect cells but very low temp kills cells by
chemicals
freezing
Nice to know!  No inflammation
Oncosis refers to cell swelling and cytoplasmic coagulation; it is Additional information from Dr.
sometimes used to describe the changes that occur before Pocheko’s lecture (2022)
necrosis. This term, however, does not appear in the 2018
recommendations of the Nomenclature Committee on Cell Death.  Stages of Apoptosis
Cell Death by Suicide (Apoptosis)  Initiation through
 Apoptosis: suicide/programmed cell death transmembrane or
intracellular signals
 Cell decided to die
 Control and integration of
 Cells are born, live for a given period of time and then die via
pro and anti-apoptotic
apoptosis
factors
 Physiological cell death
 Executioner phase – the
 Cell suicide caspases
 Cell deletion  Phagocytosis
 Programmed cell death  Clinico-pathologic
 Why should a cell commit suicide? correlations: apoptosis and
 Apoptosis is needed for proper development
 Ex. Resorption of tadpole tail, formation of fingers and C. THE APOPTOTIC PATHWAYS
toes of fetus, sloughing off of inner lining of uterus, Doc Medina showed a video about the Apoptotic Pathway. It is advised
formation of connections of neurons in the brain tthat you watch it first before reading the next parts.
 Apoptosis is needed to destroy cells
Link: https://www.youtube.com/watch?v=wREkXDiTkPs
 Ex. Cells infected with viruses, immune cells, cells with
damaged DNA, cancer cells
 Where can apoptosis be encountered?
 Growth of embryo
 Tissue homeostasis
 Immunology
 Chronic viral diseases
 Neurodegenerative diseases
 Reperfusion injury
 Insulin-dependent diabetes
 Atherosclerosis
 Myocardial infarction
 AIDS (apoptosis partly causes decline in CD4+ T-cells)
→ Development and treatment of malignancies
 What makes a cell decide to commit suicide?
 Withdrawal of positive signals (signals which allow cell to
continue to perpetuate) Figure 25. Pathway of Apoptosis.
 Examples: Growth factors for neurons, Interleukin-2 (IL-2)
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 Results in a quick and clean cell death, without damaging its

neighbors, or eliciting an immune response.
 Every cell is equipped with the “cell death pathway”.
 Intracellular proteolytic pathway
 DNA is broken into small 200bp units
 Basically:
 Cytoplasm shrinks
 Mitochondria releases cytochrome c
 Outer surface of plasma membrane gets coated with a
different sugar- one that macrophages can sense and
phagocytose
 Two pathways:
 Intrinsic pathway
 Extrinsic pathway

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Intrinsic Pathway
 Always involves mitochondria
 Can lead to caspase-dependent and caspase-independent
apoptotic pathways
 Caspase-dependent apoptosis: activation of executioner
caspases
 Caspase-independent apoptosis:
 ATP synthesis cessation
 Excess ROS production (leads to oxidative damage)
 Proteolysis
 Chromatin condensation
 DNA damage via apoptosis-inducing factor (AIF) and
endonuclease G (EndoG)

Figure 27. The Apoptotic Pathways. The extrinsic pathway will also
activate the intrinsic apoptosis (via cleavage of BID). But, intrinsic
pathway will not necessarily activate the extrinsic pathway.

Extrinsic Pathway
 Involves a cell receptor
 Trophic factors are like food for the cell
 If there are no more trophic factors, it will die
 Utilizes signals (trophic factors) that promotes its death or
(food for cells, absence causes death) from outside
 Signal from extrinsic pathway alone is not strong enough to
have apoptosis to occur
 This recruits the intrinsic pathway

Associates with Membrane integrity

Cleavage of Bid mitochondria, loss in
into tBid particularly BAX
mitochondria
and BAK 1

Caspase 3 activation Activation of

Cytochrome c
(signal caspase 9 and
attaches to apaf 1
amplification) inhibition of IAPs

Figure 26. Intrinsic pathway of apoptosis

“Just be able to demarcate what is extrinsic and what is intrinsic.

For example, the mitochondria is the most important part of the
intrinsic pathway-- that’s where it always starts.”
Note: Due to the low quality, the picture from Dr. Medina’s slides have been replaced
with the one below (similar mechanism). Original picture is in the appendix Figure 28. Diagram depicting the extrinsic pathway.

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D. CONTROL OF CELL NUMBERS AND CELL SIZE

 3 processes operate to control the eventual form a body part
takes
 Cell growth
 Cell division
 Cell death
 Single celled organisms grow as fast as they are able to limited
by factors such as food availability
 Multicellular organisms receive signals from other cells in the
body
 Mitogens – allow cell to enter cell cycle
Figure 29. Extrinsic pathway of apoptosis. FADD= FAS-associated  Growth factors – increase in cell mass
death domain  Survival factors – suppress apoptosis
 Modulates whether cell survives or undergoes death
Nuclear Dissolution
 Phase change of chromatin during apoptosis E. DEREGULATION OF APOPTOSIS IN MALIGNANCIES
 Start: heterogenous, genetically active network The following weren’t discussed by Doc Medina. These were just lifted
 End: inert, highly condensed form that is fragmented and from the 2023 trans.
packaged into apoptotic bodies
 Extrinsic pathway may activate intrinsic apoptosis but not  Caspases can be inhibited by viruses
vice versa  Apoptosis can be deregulated allowing proliferation of disease
 Karyolysis: nuclear fading – chromatin dissolution due to action and/or “immortal cells”
of DNAases & RNAases  Ras – proto-oncogene
 Pyknosis: Nuclear shrinkage – DNA condenses into shrunken  Apoptosis ↓ Tumor growth ↑
basophilic mass (necrosis)  C-myc – proto-oncogene
 Karyorrhexis: Nuclear fragmentation – Pyknotic nuclei  Caspases can be inhibited by viruses
membrane ruptures and undergoes fragmentation (necrosis)  CrmA
 Baculovirusp35
 Epstein Barr Virus BHRFI protein
 Epstein Barr Virus LMP-1 protein
 Apoptosis-related cellular proteins involved in the progression of
malignancies
 p53
 pRb
 Fas
 Mdm2
 c-myc
 c-Jun
 Bcl-2 family
 Various expression levels of apoptosis-related proteins
determine patient-specific malignancy
Figure 30. The different ways of nuclear dissolution.
 Increased Bcl-2 – poor prognosis
Caspases  Increased FasL – decreased CTL number
 Proteins which degrade other proteins are employed by  FasL induction (with doxorubicin) – determines
apoptosis chemosensitivity
 Synthesized as inactive precursors called procaspases  Overexpression of Bax – improve efficacy of chemotherapy
 These are activated by other proteins when the right signal is  p53 antibodies – resistance to chemotherapy cisplantin + 5-
received Fluorouracil
 One caspase cleaves the lamin proteins resulting in the  p53 gene status – modulates chemosensitivity
irreversible breakdown of the nuclear membrane  p53 level – predictor for response to chemo/ radiotherapy
 Ex: Caspase-1 (ICE), Caspase-2 (ICH-1, Nedd-2), Caspase-3 (advanced head and neck carcinomas, epithelial ovarian
(CPP32, Apopain, Yama), Caspase-4 (ICH-2, TX, ICEreıı) cancer)
 Substrates: PARP, DNA-PK, pRb, Lamins, NuMA, Fodrin, β-  Mutant p53 – overall shortened survival (breast cancer)
Aktin, Mdm2, Cyclin A2, Presenilin, etc.  Ratio of Bcl-2/Bax – prognostic factor (hematologic
 Caspase 3 is where extrinsic and intrinsic apoptosis converge malignancies, colon cancer)
 Bcl-2 alone – prognostic factor (advanced ovarian cancer)
“Just know that there are several caspases and they have different
substrates all leading towards cell death.” F. OTHER CELL DEATH MECHANISMS
Cell Responses to Infection
 Pyroptosis
 Autophagy
 Necrosis
 Oncosis

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**Nice to know!
In regulated necrosis mediated by RIP3, MLKL is suspected to
execute necrosis possibly via activating ion channels or forming
pores in the plasma membrane causing cell to swell and burst;
ROS formation is also suspected to produce necrosis via oxidation
of lipids (e.g., peroxidation of membrane lipids), proteins, and
nucleic acids (e.g., DNA damage), as well as activating other cell
death mechanisms (e.g., via p53 signaling)

Oncosis
 ATP-dependent
 There is swelling
 Might lead to necrosis

Figure 3`. Pyroptosis vs. Autophagy. In pyroptosis, the cells swell,

while in autophagy, the cells shrink.

Pyroptosis
 Form of programmed cell death associated with antimicrobial
responses during inflammation
 Immune cells that recognize certain danger signals within
themselves produce cytokines, swell, burst, and die
 Release a lot of internal cellular components which induce
inflammation (immunogenic)
 Can be considered a peculiar type of necroptosis (Galluzzi et al., 2014)

Autophagy
 Autophagosomes + lysosomes → autolysosomes (digest
components)
 Eats up internal contents of the cell
 Cell eventually dies since there is not enough organelles and
cellular structures to maintain life
 The cell is eaten from within (self- digestion) Figure 32. Cell Oncosis. (Ischemic cell death)
 Thus is so clean  no inflammation
 Autophagy inhibition : To differentiate if autophagy caused Cell death is more complex. More variations were not depicted in
death or it was just there during cell death; cells undergoing cell the lecture because they are not as common, the lecture only
death due to autophagy will still survive includes the major ones.
 Activated by cell stress
 Bulk cytoplasm or direct targeting
IV. REFERENCES
 Four main phases of Autophagy Medina, P. Lecture slides (2024)
 Induction and Nucleation UPCM 2023 trans. Cell cycle and cell death.
 Expansion
 Maturation
 Fusion END OF TRANS

Necrosis
 No organelles located in blebs
 Cell membrane breaks and there is no formation of blebs
 Destruction of membrane→ spilling out of contents (e.g.,
danger-associated molecular patterns) → inflammation
 Exhibits swelling
 ATP independent
 No cellular mechanism as of yet**
 Previously assumed to be a passive end-process following cell
injury or trauma

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V. APPENDIX

Figure 33. Stages of Meiosis I

Figure 34. Stages of Meiosis II

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