The cellular processes of division and checkpoint regulation

3. BLOCK 1
 INTRODUCTION TO MEDICAL GENETICS, CELL
CYCLE.
 DNA AND RNA STRUCTURES
 DNA REPLICATION
 GENE EXPRESSION: TRANSCRIPTION AND
TRANSLATION
 GENETIC CODE AND MUTATION
 REGULATION OF GENE EXPRESSION

4. BLOCK 2
 PEDIGREE REPRESENTATION/SINGLE GENE
DISORDER.
 POPULATION GENETICS
 PRENATAL SCREENING
 RECOMBINANT DNA AND DNA LIBRARY
 TECHNIQUES OF GENETIC ANALYSIS;
ELECTROPHORESIS, BLOTTING TECHNIQUES,
PCR.

5. BLOCK 3
 CYTOGENETICS: INTRO, ABNORMALITIES OF
CHROMOSOME- NUMERICAL
 STRUCTURAL ABNORMALITIES.
 TECHNIQUES OF GENETIC ANALYSIS: FISH, RFLP,
DNA FINGERPRINTING, SANGER SEQUENCING,
ALLELE SPECIFIC HYBRIDIZATION, ELISA.
 MULTIFACTORIAL INHERITANCE.

6. DR E MONDAY.

7. LEARNING OBJECTIVES
 At the end of this lecture, Student must be able to
 Describe the phases of cell cycle- 1
 Explain the role of cyclins and cyclin dependent kinase in
the cell cycle checkpoints- 3
 List tumor suppressor genes and identify diseases
associated with the Tumor suppressor genes
 Describe Labile, stable and permanent cells listing
examples of each.
 Explain the effect of chemotherapy on labile cells
 List Common chemotherapy drugs and the phase of cell
cycle they act on.
 Define and describe LiFraumeni syndrome.

8.  Stages through which a cell passes from one cell
division to the next. It is the stage in which a cell grows
and prepares for division. When the cell divides it
divides into 2 equal identical cell.
 Cells are the smallest, functional unit of an organism.

11.  The study of chromosome their structure, and
inheritance is called Cytogenetics. Chromosomal
analysis have become an important diagnostic
procedure in clinical medicine.
 And can be used in clincial diagnosis.
 Gene mapping
 Cancer cytogenetics
 Prenatal diagnosis.

12. CHROMOSOMES
 Chromosomes are thread-like structures located
inside the nucleus of animal and plant cells. Each
 chromosome is made of protein and a single
molecule of deoxyribonucleic acid (DNA). Passed from
parents to offspring, DNA contains the specific
instructions that make each type of living creature
unique.

13. CELL CYCLE.

14.  Mitosis is obviously crucial for growth and
differentiation, but it takes up only a small part of the
life cycle of a cell. The period between two successive
mitoses is called interphase, the state in which most
of the life of a cell is spent.
 Immediately after mitosis, the cell enters a phase,
called G1, in which there is no DNA synthesis.

15. Cell Cycle
Interphase
(Growth)
M phase
(Mitosis)
G1 (growth)
S (synthesis)
G2 (growth)
G0
(resting)
Richard Wheeler (Zephyris) 2006

16. Cell Cycle
• G1 phase
• Synthesis of proteins, organelles
• Length varies depending on conditions
• Mitogens:
• Extracellular signaling molecules, usually proteins
• Stimulate cell division
• Function via cyclin dependent kinases (Cdks)
• Growth factor: Stimulates growth in size
• Some molecules both mitogens and GFs
• Terms sometimes used interchangably

17. S-PHASE.

18. Cell Cycle
• S phase
• Synthesis of DNA
• Chromosomes 
two sister chromatids
• G2 phase
• Growth in preparation for mitosis

19.  During G2, the cell makes final preparations for
division of the nucleus and cytoplasm. A key event
that continues from S into G2 is the error correction in
DNA repair. The checkpoint between G2 and mitosis
or meiosis (M) is not passed until repair activities have
been completed.

20. G0 Phase
• May occur in absence of mitogen stimulation
• Specialized non-dividing state
• Most cells in our body are in G0
• Some permanent G0
• Others go in/out

21. G0 Phase
• Neurons, skeletal muscle cells
• Permanent G0 state (“terminally differentiated”)
• Liver cells
• Often in G0 but may divide if stimulated
• Fibroblasts, lymphocytes
• Enter and exit G0 many times in their lifespan

22. G0 Phase
• Bone marrow cells, GI epithelial cells, hair follicles
• “Labile cells”
• Rapidly dividing
• Rarely/never enter G0
• Most effected by many forms of chemotherapy

23. Mitosis
• Shortest (most rapid) portion of cell cycle
• Divided into phases
• Prophase
• Prometaphase
• Metaphase
• Anaphase
• Telophase
Richard Wheeler (Zephyris) 2006

24. Mitosis
Prophase
• Chromosomes condense
• Spindle fibers forms
Ali Zifan/Wikipedia

25. Mitosis
Prometaphase
• Chromosomes organize
on mitotic spindle
Ali Zifan/Wikipedia

26. Mitosis
Metaphase
• Chromosomes line up on
metaphase plate
Ali Zifan/Wikipedia

27. Mitosis
Anaphase
• Chromosomes separate
Ali Zifan/Wikipedia

28. Mitosis
Telophase/Cytokinesis
• Spindle breaks down
• Cell divides
Ali Zifan/Wikipedia

30. Cell Cycle Control
• Cells regulate progression through “checkpoints”
• Also called “restriction points”
• G1-S (prior to S phase entry)
• G2-M (prior to mitosis)
• M phase (prior to anaphase/cytokinesis)
• Arrests cell if conditions not appropriate
• First checkpoint: Late G1 (G1-S)
• Cell commits to cell cycle/growth
Richard Wheeler (Zephyris) 2006

31.  The progression of a cell through the cell division cycle is
regulated at a number of checkpoints by a wide array of
genes.
 In the first phase, G1, preparations are made to replicate the
genetic material. The cell stops before entering the DNA
synthesis phase, or S phase, to take inventory.
 Are we ready to replicate our DNA?
 Is the DNA repair machinery in place to fix any mutations
that are detected?
 Are the DNA replicating enzymes available? Is there an
adequate supply of nucleotides?
 Is there sufficient energy?

32. Cell Cycle Control System
 Regulation of the cell cycle is a crucial process to the
survival of the cell. Cell regulation includes the
detection and repair of genetic damage and also
prevention of uncontrolled cell division. The
molecular events that control the cell cycle occurs in a
sequential fashion and is impossible to reverse the
cycle. This process of cell cycle regulation is done by
cyclins, CDKs, and tumor suppressors.

33. Cell Cycle Control
• Cyclin Dependent Kinases (Cdks)
• Central components of cell cycle control
• Kinase enzymes (lead to phosphorylation of other proteins)
• Always present in cells but inactive
• Depend on cyclins to activate
• Cyclins: regulatory proteins – activate Cdks
• Cyclin-Cdk complexes
• Phosphorylate regulatory proteins
• Allow progression through cell cycle

34.  The main brake on the process is the retinoblastoma
protein, Rb. When the cell determines that it is
prepared to move ahead, sequential activation of
cyclin-dependent kinases (CDKs) results in the
inactivation of the brake, Rb, by phosphorylation.
Phosphorylated Rb releases the S phase–regulating
transcription factor, E2F/DP1, and genes required for S
phase progression are expressed.
 phosphorylation inactivates Rb

35.  Proteins like the cyclin dpendent kinase, kinases and
cyclins control the switches for the cell cycle causing
the cell to move from G1 to S or G2 to M. .
 Cyclin and Cdk1 complexes drive the transition
between G2 phase and M phase.
 Regulatory molecules are of two classes
 cyclins
 cyclin-dependent kinases.

36.  A cyclin forms a complex with Cdk, which begins to
activate the Cdk, but the complete activation requires
phosphorylation, as well. Complex formation results in
activation of the Cdk active site. Cyclins themselves
have no enzymatic activity but have binding sites for
some substrates and target the Cdks to specific
subcellular locations.

37.  If the cell determines that it is unready to move ahead
with DNA replication, a number of inhibitors are
capable of blocking the action of the CDKs, including
p21Cip2/Waf1, p16Ink4a, and p27Kip1

38. G1-S Checkpoint
Active Cyclin
Cdks
P
P
P
E2F
Inactive E2F
Cell Growth

39. G1-S Checkpoint
• Cyclin-Cdk complexes activate E2F proteins
• Transcription factors
• Bind to DNA promoter regions
• Activate genes for S phase
• E2F normally inhibited
• Inhibited by E2F binding to retinoblastoma proteins (Rb)
• Inhibition released by G1-S-Cdk phosphorylation of Rb
• Rb regulates cell growth
• “Tumor suppressor”

40. G1-S Checkpoint
• DNA damage can arrest cell division
• Allows for repair
• Prevents development of mutant cells/cancer
• DNA damage initiates signaling pathways

41. G1-S Checkpoint
• ATM pathway: Activated by double strand breaks
• ATM: Ataxia Telangiectasia Mutated
• ATM gene mutation 
Ataxia Telangiectasia
• ATR pathway: Single stranded breaks
• Both lead to phosphorylation of proteins
• Causes cell cycle/growth arrest

42. P53 Protein
• Major target of ATM/ATR systems
• Phosphorylated after DNA damage
• Prevents p53 breakdown
• Increases levels/activity
• p53 induces transcription of p21 protein
• p21 binds to Cdks 
inhibits Cdk activity
• Blocks cell progression through cell cycle
• p53/p21 = tumor suppressors

43. P53 Protein
DNA Damage
P53
Unstable Protein
Rapid Breakdown
P53
Stable
CdkInhibition
Growth Arrest
P21
P53
P21 gene

44.  Cell cycle checkpoints are the regulatory pathways that
control the order and the timing of the transitions of
the cell cycle. The checkpoints also ensures that
critical events such as replication of DNA and
segregation of chromosomes are completed before the
cell progresses further through the cycle.

45.  The cell-cycle checkpoints respond to the cellular
damage by slowing the cycle to provide time for repair
and it also induces transcription of genes that facilitate
the repair. The loss of the checkpoints results in
instability of chromosomes and it can result in the
transformation of normal cells into cancer cells.

46.  At the end of S phase, when the cell has exactly duplicated its
DNA content, a second inventory is taken at the S checkpoint.
 Have all of the chromosomes been fully duplicated?
 Were any segments of DNA copied more than once?
 Do we have the right number of chromosomes and the right
amount of DNA?
 If so, the cell proceeds to G2, in which the cell prepares for
division by synthesizing mitotic spindle and other proteins
needed to produce two daughter cells.
 When DNA damage is detected, the p53 pathway is normally
activated. Called the guardian of the genome, p53 is a
transcription factor that is normally present in the cell in very
low levels.

47.  Normally, p53 is bound to mdm2, a ubiquitin ligase,
that both inhibits p53 transcriptional activation and
also targets p53 for degradation in the proteasome.
When damage is sensed, the ATM (ataxia-
telangiectasia mutated) pathway is activated; ATM
phosphorylates mdm2, which no longer binds to p53,
and p53 then stops cell cycle progression, directs the
synthesis of repair enzymes, or if the damage is too
great, initiates apoptosis of the cell to prevent the
propagation of a damaged cell

48.  Inducers of p53 include hypoxemia, DNA damage
(caused by ultraviolet radiation, gamma irradiation, or
chemotherapy), ribonucleotide depletion, and
telomere shortening

49.  Maturation-promoting factor. MPFs activate other
proteins through phosphorylation. These
phosphorylated proteins, in turn, are responsible for
specific events during cycle division such
as microtubule formation and chromatin remodeling.
 MPF must be activated in order for the cell to
transition from G2 to M phase.

50.  MPF is disassembled in a negative feedback loop. In
intact cells, cyclin degradation begins shortly after the
onset of anaphase (late anaphase), the period of
mitosis when sister chromatids are separated and
pulled toward opposite spindle poles.

51.  The genes encoding cyclins and CDKs are conserved
among all eukaryotes. Proteins like the p27 and p53
and Rb. prevent the cells from passing check points.
They are also known as protein suppressors. P27
protein binds to cyclin and CDK blocking the entry
into the S phase.

52.  P53 protein blocks the cell cycle at the M checkpoint if
the DNA is damaged.
 Binds DNA directly and produces proteins that stops
the progression of cell cycle.
 P53 mutation is the most frequent mutation found in
cancer cells.
 P53 fucntions by blocking the cycle giving the cell time
to repair its DNA. If there is severe damage in the DNA
the protein causes the cell to apoptosis.

53.  Abnormalities in TP53 Ž
multiple malignancies at an
early age. Also known as SBLA cancer syndrome
(sarcoma, breast, leukemia, adrenal gland).
Li-Fraumeni syndrome

54. OTHER TUMOR SUPPRESOR GENE

55.  Bleomycin blocks G2 phase:
 Mechanism: Induces free radical formation breaks in DNA
strands.
 Uses: Testicular cancer, Hodgkin lymphoma.
 Methotrexate: Folic acid analog that competitively
inhibits dihydrofolate reductase decreaces dTMP and DNA
synthesis.
 USES:Cancers: leukemias (ALL), lymphomas,
choriocarcinoma, sarcomas.Non-neoplastic: ectopic
pregnancy, medical abortion (with misoprostol),
rheumatoid arthritis, psoriasis, IBD, vasculitis.

56. M PHASE INHIBITORS

57. Retinoblastoma
• Rare childhood eye malignancy
• Mutations in RB1 gene
• Codes for Rb protein
• Abnormal Rb 
Unregulated cell growth (via E2F)
Wikipedia/Public Domain

58.  Cell types:
 The cells that have stopped dividing temporarily or
reversibly are said to be in the state of quiescence called
G0 phase
 Permanent: Remain in Go phase regenerate from stem
cells, eg neurons, skeletal mzle, cardiac muscle, RBC.
 Stable: enter G1 from Go when stimulated: hepatocytes,
lymphocytes, renal tubular cells, parenchymal cells of
many glands, and numerous mesenchymal cells (e.g.,
smooth muscle, cartilage, connective tissue,
 endothelium, osteoblasts
 Labile: never go to Go phase they are rapidly dividing. Eg
bone marrow, hair follicles, skin, gut epithelium.

61.  Which of the following phases of cell cycle does
methotrexate block
 A. G1
 B. G2
 C. S
 D. M-phase
 .

62.  A 52 year old was diagnosed with hodgkin lymphoma,
the ocologist decided to start him on a drug the target
metaphase of the diving cells, which of the following
medication was he presecribed
 A. bleomycin
 B. 5-Flucytosine
 C. Vinblastin
 D. Paclitaxel
 E. Methotraxate

63.  An oncologist is reviewing a patient who was on
remission for the past 5 years but now has a relapse of
the cancer, the medication used targets stable cells
which of the following cells will be affected?
 A. Hepatocytes
 B. Bone marrow cells
 C. Osteoblasts
 D. Hair follicles
 E. Gut Epithelial cells
 F. Cardiac myocytes

64.  A patient with known mutation involving Rb gene
should be screened for which cancers?
 A 27 year old female is concerned about cancers in the
family,, her mother and elder sister recently had a
mastectomy due to stage 1 breast cancer, she lost her
younger brother 7 years ago to stage 4 Leukemia and
her uncle is also on remission from bone cancer.
 Which of the following genes mutation is likely
implicated in this family’s cancer?
 Which other organ should she be screened for cancer?