2/11/2010 20

Isma’eel
Batool
CyclinsAl-Saify
and
Matalqa
14
Retinoblastoma gene
 Cyclins and Retinoblastoma gene
Today , We will try to finish the repair ..
On Sunday morning we will discuss the marks!

Just to remind you .. You remember probably the Cyclins and last lecture we said that they are the family
of proteins that control the entry of the cells at specific stages of cell cycle.
And probably we mentioned some examples of these Cyclins and we said in order to accomplish their
function, they have to bind to Cyclin-dependent kinases (CDKs) .. And these different combinations are
essential for each phase of the cell cycle, usually they exert their effect by phosphorylating certain
proteins (kinase phosphorylate proteins). and we mentioned the examples and I think we stopped here
slide 22 from ( patho slide #7 tissue repair #1 ) .

Retinoblastoma Gene 
Retinoblastoma (Rb) was the first tumor suppressor gene described in science  it's a good example
actually to know the relationships between genes (in general) and retinoblastoma genes which are key
products that interact with each other .
 this is one of the most important steps regulating the recognition from G1 (Gap 1) into S phase (from the
presynthetic to the synthetic phase) and this is the most important gene products which control this phase.

So we will try to highlight the main functions of

this retinoblastoma genes and then we will see
the relationships between cyclins and CDKs .
(Patho slide #8 .. tissue repair #2 slide #1).

ž  Hypophosphorylated retinoblastoma
(RB) , forms a complex with the specific
transcription factor (E2F) and the DP1 
leading to blocking the effect of E2F  The E2F
main function is to activate and proceed in the
cell cycle so such formation of the
hypophosphodepression acting or the
hypophosphorylated form will block this step,
and this blocking is mediated by the histone
deacetylase causing chromatin compaction
(remodeling) and then CyclinD/CDK4, and
cyclinE/CDK2 phosphorylate RB 
Phosphorylated RB dissociated from the
complex, leading to activation of E2F. (and I
think this is better illustrated in the second picture).

NOTE: Target genes for E2F include: cyclin E, DNA

polymerase, thymidine kinase, dihydrofolate
reductase, and others.

.. LOOK @ THIS FIGURE

ž The cycle on the right lower corner 

transmission from G1 to S phase there are
complexes of the CDKs and if this proceeds in
this way it will lead to inactivation of the
hypophosphorylated RB (active form) leading to
the formation of hyperphosphorylated RB (inactive form) and dissociation of the E2F transcription factor and then
S phase genes and transcriptional activation will proceed .
 the presence of hypophosphorylated RB (look @ the figure) will lead to transcriptional block and therefore
blocking the S phase genes .
 this is nonchromatin - in association with other chromatin remodeling factor - and they are the Histone
deacetylases and Histone methyltransferases which facilitate the process of inactivation or blocking the
transcriptional effect of this phase .

ž  El upper cycle in the figure (previous page)  there are two components here ;
1. we can get the growth factors ( EGF , PDGF ) which will activate the process from G1 and therefore
creating more E2F sites or more transcriptional activity and proceeding to the S phase.
2. On the other hand we can have some other growth inhibitors (TGF-B (transforming growth factor beta),
p53 , others) which exert their effect through the CDK inhibitors (e.g. the p16 (INK4a)) . and this will lead
to inactivation of cyclins D/CDK4,6 and cyclins E/CDK2 and then blocking this process .
ž NOTE: we have cyclins , CDKs and CDK inhibitors
ž
 So any growth of the body whether it was part of a normal growth or an abnormal growth  is
regulated by the balance between these two equilibriums; growth factors & growth inhibitors ..
activation or inactivation of RB, and I think you will learn more about this in the neoplasia.

Rb and cell cycle regulation (the same topic) 

ž

You can see here the activation ,

inactivation and the
hypophosphorylated Rb  which is
the active form , the
hyperphosphorylated Rb  which is
the inactive form and this is just to
show you the balance in neoplasia we
have here what we call them
ONCOGENIC VIRUSES (Viruses which
cause neoplasia "tumors" ) …
 and there are good examples of
these like the HPV (human pavilloma
viruse) .. and we have the E7 protein or
the SV40-T antigen which also exert
their effect on the retinoblastoma
leading to the formation of malignancy .

 So if you want to understand neoplasia .. if you want to understand repair .. or

understand pathology in general .. First you need to understand the cell cycle which is
the basis of every disease .. which is the basis of every pathology at the cellular level .. So
we have what we call the cellular biology .. the molecular biology .. to understand the
different steps in initiation of neoplasia or any abnormal disease …

BACK to the Cyclins 

you know now that we have some CDK inhibitors which will block the effect of CDKs compounds or complexes
and they regulate of course the cell cycle at the checkpoints.
The main checkpoints (G1-S phase and G2-M phase)  There are two main families :
1. Cip/Kip family  p21, p27 and p57
2. INK4/ARF family  p16INK4A, p14ARF
ž
We are discovering more and more and more of these inhibitors .. every week .. every
month they are discovering some new cyclin–dependant kinase inhibitors .. these are
just some examples of CDK inhibitors .

P53 (Gaurdian of the Genome) and the cell cycle 

P53 is one of the most important tumor suppressor genes (although it was discovered a little bit later than
retinoblastoma genes).

ž
LOOK @ THIS FIGURE .. (role of p53)

If mutation occurs  TP53 is stabilized (normal p53 gets

activated by DNA- damaging agents or hypoxia)
TP53  induce p21 (CDKN1A) transcription
P21  is a CDK inhibitor, thus arresting the cell at G1 until DNA
is repaired

If a DNA damage happened or hypoxia or any insult leading to

p53 activation and binding to DNA  this will lead to
transcriptional up-regulating of target genes leading to
abnormal transcription. So in this case there will be some sort
of activation of p21 which is a CDK inhibitor  which will lead to
arrest of these abnormal DNA at the G1 phase.

NOTE: Also there is another gene which is responsible for the

DNA repair  which is called the GADD45
ž
Such arrest of these cells at the G1 phase will allow them to do
the DNA repair successfully and then the cells will get back to
normal but if such repair fails it will lead to the activation of
other genes and one of the most important of these genes is
the BAX genes which lead to apoptosis  will lead to
destruction of this abnormal cell .

 And this is why the p53 or TP53 is called the guardian of the genome because if you don’t
have these control mechanisms this will lead to the presence of a lot of mutations and
accumulation of these mutations will lead abnormalities, tumors and diseases ..

NOTE: The new name of p53 is TP53 but the better name is P53.

Signals for Cell Growth and Differentiation 

There are some soluble polypeptide growth factors and there are few Insoluble elements of ECM
interacting with integrins .. so we'll be discussing some of these growth factors ..
 They are Chemical mediators that affect cell growth by binding to specific receptors on the cell surface or
intracellularly, and they are the most important mediators affecting the cell growth.
ž
They are present in serum or produced locally. and they exert pleiotropic effects; this means that they
can do different actions , they can induce proliferation, they can direct the cell migration, they can lead to
differentiation, or tissue remodeling.
ž
 Also they regulate the growth of cells by controlling the expression of genes that regulate the cell
proliferation , these genes that control cell proliferation are called (protooncogenes); when they get mutated 
 these protooncogenes will become oncogenic (if there is any defect of these protooncogenes).

 Some examples of these growth factors :

1. EGF (epidermal growth factor) & TGF-a (transforming growth factor)
ž - Binds to its receptor ERB B1 (specific receptors)
ž - Mitogenic for epithelial cells & fibroblasts; which means they induce the cooperation of epithelial cells &
fibroblasts and of course migration of epithelial cells.

2. PDGF (platelet-derived growth factor)

ž - Migration & proliferation of fibroblast, smooth muscle cell & monocyte; it acts as a chemotactic factor.

3. FGFs (fibroblast growth factors)

ž - Mitogenic for fibroblast & epithelial cells; angiogenesis; chemotactic for fibroblasts
ž - they play essential role in Wound healing.

4. VEGF (vascular endothelial growth factor)

ž - It's main role is Angiogenesis
ž - It Increases the vascular permeability

5. HGF/scatter factor (hepatocyte growth factor)

ž - Mitogenic to most epithelial cells including hepatocytes.
ž - Promotes scattering and migration of cells.

Transforming Growth Factor Beta (TGF-b) 

we mentioned the transforming growth factor alpha (TGF-a)

a. One of the most important growth factors is Transforming Growth Factor Beta (TGF-b) …
b. TGF-b binds to 2 receptors (types I &II) with serine/threonine kinase activity
c. The receptors phosphorylate the cytoplasmic transcription factors called Smads and we will see
them in the diagram later, these Smads will enter the nucleus and associate with other DNA
binding proteins activating or inhibiting gene transcription according to the receptor which
activates it and also according to the concentration of the TGF-b.
d. They act like Inhibitors of most epithelial cells and leukocytes. And they increase the expression
of cell cycle inhibitors (Cip/Kip, INK4/ARF).
e. They stimulate proliferation of fibroblasts & smooth
muscle cells.
f. They induce fibrosis (fibroblast chemotaxis, production
of ECM, ↓ proteases, ↑ protease inhibitors).
g. They have strong anti-inflammatory effect.

TGF-b Signaling 

.. LOOK @ THIS FIGURE

The role for the cytoplasmic form of promyelocytic

leukaemia protein (cPML)  The activation of these cPML will lead to
the activation of the transcription growth factors within the
nucleus .. At the cell surface, cPML might interact with the two TGF-
receptors (TRI and TRII) and act as a bridging factor between SARA
and Smad2/3.
 Upon stimulation with TGF-b, cPML  promotes the transfer of the complex containing TRI, TRII, SARA
and Smad2/3 into early endosomes  There, cPML might dissociate from the complex (a),   allowing
Smad2/3 to interact with SARA (b) and to be phosphorylated (c) by TRI  Phosphorylated Smad2/3 moves into
the nucleus to propagate TGF-b signaling.

Patterns of Intercellular Signaling 

How do these growth factors exert their effect ?

 There are three main ways of intercellular
signaling between the different cells ;
ž
1- One of them we call it the Autocrine signaling
where we have the signal which is released from the
cell and exerts its effect on surface receptors which
are present on the same cell  So this is more like
autonomous Activation we call it autocrine.

2- The paracrine signaling  we have cells which

secrete these extracellular signals to the affecting
adjacent target cells.
ž
3- Endocrine signaling  if we have extracellular
signals produced on hormones in one area and then
secreted to the blood and acting it's effect on other
distant target cells (like the effect of the thyroid
gland).

.. LOOK @ THIS FIGURE

Receptors for Growth Factors 

The main four groups are :
ž 1- Receptors with intrinsic tyrosine kinase activity
ž 2- Receptors lacking intrinsic tyrosine kinase activity that recruit kinases
ž 3- Seven transmembrane G-protein coupled receptors
ž 4- Steroid hormone receptors

Examples of Signal Transduction

Systems 

.. LOOK @ THIS FIGURE

This diagram illustrates the different types

of these receptors, so we have different
receptors .. with or without kinase
activities .. all the cells try to bring G-
protein kinase receptors on the other
cytokine or endocrine or steroid receptors.
Signals from Tyrosine Kinase Receptors 
And this diagram  to show you the
growth factor ligand and the
Receptor-ligand complex activating
the different transcription factors.

Extracellular Matrix (ECM) 

 ECM : Component important in cell signaling.
 A major component of all tissues
 provides the backbone & support for the body, imagine the human being without the ECM it is just like a ball of
meat because it's the integral part of the bone , the cartilage and the connective tissue.. so it is the backbone.
 regulates cell growth, movement and differentiation.
ž
And very frequently you will hear about the ECM – cell interactions or cell – cell interactions and ECM
interactions. Which are very important in health and in diseases  they are very important in healthy individuals
as well as in diseased individuals  they have very important role in inflammation, repair, neoplasia,
autoimmune diseases and in any other diseases affecting the human body.

 Their main functions are :

1. Mechanical support
2. Control of cell growth
3. Maintenance of cell differentiation
4. Scaffolding for tissue renewal
5. Storage place and presentation of regulatory molecules in the interactions
6. Tissue microenvironments

NOTE: we mentioned last time the liver as an example, we said if there is an ECM there will be an easy
regulation of the hepatocytes but if the ECM is lost then the repair will be by fibrous tissues and there is
a big difference between regeneration by the original native cells or repair by fibrous tissue which will
.impair the function and the structure of the organ

Major Components of the ECM 

1- Basement membrane: is the basement membrane of any cell present in the body;
and it's composed mainly of 

 Type IV collagen (there are different kinds of collagen from 1 to 12 and the type 4 is the most
important in basement membranes where as type 1,2,3,5 are main components of the interstitial
matrix which we call them fibrillar and non-fibrillar collagens).
 Proteoglycans.
 Laminin (Adhesive glycoprotein).
 2- Interstitial matrix:
 Fibrillar and nonfibrillar collagens other than type 4 collagen.
 Elastin : from its name which is a fiber that maintains elasticity in the tissue and some sort of
compressibility of the tissue.
 Proteoglycans
 Fibronectin (Adhesive glycoprotein).

ž The basement membrane is a mesh like complex structure.

ž You can see the relationship between these interstitial matrix components and the other components.
 Also you can see the signaling processes; the exchange of signals between the different parts of the ECM
and the basement membrane material.

Components of the ECM 

ž1- Collagen :
 The most common protein in animals
 They divide into Fibrillar & non-fibrillar
 They need to get hydroxylation because  they are present in their inactive form so they will need to be
activated so they need hydroxylation, and this is mediated by vitamin C, and that’s why vitamin C is
important in healing providing strength (el nas yalli be3malo 3amaliyyat advised to take vitamin C ,this will
potentiate the effect of the hydroxylation of these collagens and this will lead to the proper healing, wel nas
yalli 3ndhum el es8arbou6 (vitamin C deficiency) they get these abnormality in their skin and their mucosa
that's mainly because of the deficiency of collagen).
 Fibrillar collagens form most of CT in wounds & scars.
 Non-fibrillar (type IV) main component of basement membrane (BM).
 Genetic defects: Ehlers-Danlos syndrome & Osteogenesis imperfect  which are diseases of connective
tissue and diseases in the collagen fibers. Those with Osteogenesis imperfect  they have under-
developed bone, under-developed soft tissue and this will lead to frequent fracture.
ž2- Elastin :
 Provides elasticity
 Surrounded by a mesh-like network of fibrillin which supports elastin deposition.
 One of the major components of blood vessels "specially the arteries and the aorta" is Elastin ; Elsatica
interna , Elsatica externa. The main function of this is to maintain the elasticity because these are major
blood vessels.
 Defective fibrillin leads to Marfan syndrome  it has a physical syndromatic picture; one of the defects
is the abnormality in the aorta and blood vessels ; in addition to the other dysmorphic features of the
patient.

ž3- Proteoglycans :
 Form highly hydrated gel like material  they are water soluble.
 Protein core with many attached long polysaccharides (glycosaminoglycans)
 Act as a reservoir for bFGF (beta fibroblast growth factor).
 Integral cell membrane proteins (e.g. Syndecan)

ž4- Adhesive glycoproteins :

 there are two examples of these :
A. Fibronectin :
 – Domains bind collagen, elastin, proteoglycans, etc.
 – Binds to integrins via RGD domains
B. Minin :
 – Connects cells to collagen and heparan sulfate.

Proteoglycan 

.. LOOK @ THIS FIGURE

You see in the figure the heparin sulfate proteoglycan in

matrix. Where the heparin sulfate have the specific receptors ,
and you can see there the Syndecan which exert its effect on
the actin cytoskeleton and this will lead to migration of the
cell within the ECM.

Fibronectin 

.. LOOK @ THIS FIGURE

You see in this figure  there are different binding

sites of motifs. There are some collagen-binding
domains, fibrin-binding domains and heparin-binding
domains.
Laminin 
So you see they are large molecules with
complicated structures exerting specific and
important functions within the cells.

.. LOOK @ THIS FIGURE

Interaction Between GF, ECM and Cells 

 Once there is sort of activation through the growth factor receptors, what we call the soluble signals
and this will mediate their effect through the pathways we just mentioned to do the transcriptional
activities.

 Whereas these non-soluble signals exerting their effect directly through integrin receptors activated by
different molecules. And this will lead to focal adhesion complexes with actin cytoskeleton activation ..
these what we call cytoskeletal-mediated signals, so they are mediated by the cytoskeletal elements
within the cytoplasm rather than through the other pathways that we mentioned and this will lead to
 activation the effect of proliferation, differentiation, protein synthesis, attachment, migration and
shape change.

Repair by Regeneration 
 Replacing injured tissue by same type of original tissue cells.
 Labile & stable cells
 Involves two tissue components:
1- Cellular proliferation, regulated by growth factors & growth inhibitors.
2- Extracellular matrix (ECM) & cell-matrix interaction
 An intact basement membrane directs epithelial cell polarity & is essential for its orderly regeneration.

The End
Done by : Batool Al-Saify

Thanks to all my friends … Especially :

Raneem Wardat
Nouran Tubaishat
Rana Haddad
Haya Mesmar
Dana Halawa
Sura Al-Hami
Bayan Abu Khalil
Basma Deeb
Rasha Ebbini
Leen Shawaheen
Lubna Bataineh
Raghad Al-Shiyab
Hadeel Kofahy
Fatima Kofahy
Sarah Al-Zubi
Zeina Majthoub
Samah Rjoub
Nour Rousan
Ansam Daoud
Hanna Ighraiz
Duaa Herzallah
Samah Abu Omar
Dalia Rawashdeh
Sarah Abu Dalu
We el kul :D:D
Special thanks to my brother Bashar !