Department of Biochemistry

Faculty of Pharmacy

Al-Azhar University

Lecture No. 7

Hormones

Cell biology represents the study of the normal

physiological processes that occur in the cell

including cell cycle, differentiation, apoptosis,

angiogenesis and growth factors.

Cell Biology

It is a series of distinct biochemical and physiological events

occurring during cell replication.

Time of cell cycle is variable:

Some adult cells don’t divide (i.e. reach to cell cycle

stop phase or G0): e.g. Nerve cells and eye lens

Cell cycle

1) G1, Growing or Gap phase 1

2) S or Synthetic phase

3) G2, Growing or Gap phase 2

4) M or Mitosis phase

Stages of normal cell cycle

During this phase:

• Growth stimulatory and replication signals arrive

and accumulate within the cell.

• The cell grows in size and stores the materials

required for DNA replication.

• Cell checks everything OK for DNA replication.

G1 may be the longest phase of cell cycle.

G1, Growing or Gap phase 1

During S phase, cell replicates its DNA and doubles the

number of chromosomes.

DNA synthesis occur during S phase because:

1) Cell contains excess quantities of DNA polymerase than

during the other phases.

2) There is excess activity of enzymes that responsible for the

formation of the substrates for DNA synthesis i.e. enzymes

for formation of dATP, dGTP , dCTP and dTTP.

S or Synthetic phase

During this phase the cell prepare itself for mitosis.

Accumulates proteins that activate cell division.

Cell checks everything is OK for cell division

G2, Growing or Gap phase 2

During this phase the cell undergoes actual cell division into

two daughter cells.

It is the shortest phase of cell cycle.

Divided into six phases:

» Prophase

» Prometaphase

» Metaphase

» Anaphase

» Telophase

» Cytokinesis

M or Mitosis phase

Cell cycle is regulated by a gene that produces protein

called cyclin.

Cyclin govern the transition from one phase of the cell cycle

to another through controlling a number of enzymes called

cycline-dependent protein kinases (cdpk).

These enzymes are activated by mitogenic growth factors.

Regulation of cell cycle

The sequential events of the cell cycle are directed by a

distinct cell cycle control system, which is similar to a clock

The clock has specific checkpoints where the cell cycle

stops until a go-ahead signal is received

For many cells, the G1 checkpoint seems to be the most

important one

Regulation of cell cycle

checkpoint1G

1G

S

M

M checkpoint

checkpoint2G

2G

Control

system

1G

checkpoint1G

1G

0G

If a cell receives a go-ahead the checkpoint, 1signal at the G

cell continues on in the cell cycle.

If a cell does not receive a go-checkpoint, 1ahead signal at the G

the cell exits the cell cycle and dividing state.-a non, 0goes into G

During cancer development there is no control on the cell

cycle and lead to continuous uncontrolled cell division

without repairing of DNA leading to DNA mutation.

On time these mutations lead to more and more aggressive

tumors.

So, some strategies in cancer therapy depend on the use of

chemicals that interfere with cell cycle progression.

Cell cycle and cancer

It means the specialization of unspecialized embryonic cells

into specific cells to perform specific function.

N.B.: Normal cells are called differentiated cells but cancer

cells are called dedifferentiated cells.

Stem cells: these cells that can be differentiated into more

than one type of cells.

Application of stem cells:

• Gene therapy.

• Tissue transplantation.

Differentiation

It is also called programmed cell death

Apoptosis is a normal physiological process

occurring in damaged cells.

The term “apoptosis” is a Greek word = “Falling off”

of leaves from a tree

Apoptosis

Biomedical Importance:

• It serves as the mechanism that eliminate the damaged cells

when they are no longer needed.

• It plays an important role in embryogenesis and normal

tissue homeostasis.

• It is involved in certain pathological conditions such as

malignancy and AIDS.

• General Mechanism:

• Apoptosis is a multi-step process involve fur distinct phases:

1) Initiation phase: it start by an apoptotic stimulus, and is subject to very

complex regulation.

2) Effector phase: It occurs when pro-apoptotic signals override the anti-

apoptotic signals (the cell becomes committed to die).

3) Degradation phase: include the following:

• Shrinkage of plasma membrane and cytoplasmic organelles

• Nuclear chromatin condensation.

4) Engulfment phase: apoptotic bodies are rapidly ingested by neighboring

cells, without inflammation

N.B.: Defects in the regulation at any of these phases may ultimately

result in disease.

• Biochemistry of Apoptosis:

• Ultimately, apoptosis is the net result of the balance

between pro- and anti-apoptotic stimuli.

• The most important regulatory mechanisms of apoptosis in

mammalian cells belong to the following categories:

1) Death receptors.

2) Caspases

3) Mitochondria

4) The Bcl-2 family of proto-oncogenes

5) Certain tumor-suppressor genes

Site: may be membrane-bound or in a soluble form.

Function: act as sensors to extracellular apoptotic stimuli.

Mechanism: when apoptotic stimuli bind to specific death

receptor it lead to intracellular proteolytic cascade by

agroup of enzymes called “caspases” resulting in cellular

death within hours.

Examples: The prototypical death receptors include Fas

and tumor necrosis factor receptors (TNF-Rs).

1- Death Receptors

They belong to the family of cysteine proteases

Site and Function: They exist in the cytoplasm as

proenzymes (less active), but once activated, play important

roles in the initiation and effector phases of apoptosis.

2- Caspases

Structure: Each caspase consists of

a structurally related molecule with a

prodomain, and a large (~20 kD) and

small (~10kD) subunit that combine

to form tetramers after activation.

Classification:

• More than 13 caspase member are known,11 of which have

human counterparts.

• They are classified according to their substrate

specificity and function into:

1) Upstream enzymes [caspases 8, 9,10], that serve to initiate

and amplify the death signal

2) CED-3-like [caspases 2, 3, 6, 7], that are involved in the rapid

cleavage of vital cellular components, e.g, nuclear membrane

3) ICE-like [caspases 1, 4, 5 and 13], that are less involved in

cell death.

Mechanism:

• Like the clotting cascade, the caspase cascade proceeds in

an auto-catalytic manner, leading to intense amplification of

the initial apoptotic stimuli.

• This cascade is regulated by various cofactors (e.g., apoptotic

protease activating factor-1 and inhibitors) at the post-

translation level, by protein-protein interactions

It plays a central role in apoptosis

Mechanism: disruption of mitochondrial membrane lead to

matrix swelling and loss of mitochondrial transmembrane

potential leading to:

1. Release of specific proteins, such as cytochrome c, which

activates procaspase-9 thus activating the caspase cascade

2. Disruption of electron transport (an early feature of apoptosis)

3. Modification of cellular oxidation-reduction potential.

3- Mitochondria

Classification: Many proto-oncogenes are involved in

maintaining cell survival and proliferation.

It could promote:

A. Cell survival e.g. Bcl-2 (anti-apoptotic)

B. Cell death e.g. Bax (apoptotic)

Bcl-2

It is a membrane-associated protein located at the

mitochondrial and peri-nuclear membranes

Its role is to promote cell survival and proliferation.

4- Proto-Oncogenes (Bcl-2 Family )

• p53 is one of the most important tumor-suppressor genes

• It can induce cell death in response to DNA damage.

Defect: Mutations causing its loss of function are associated with

many human cancers.

Mechanism: activated p53 lead to cycle arrest causing either:

1) Allow DNA repair (if failed….)

2) Cell death

by initiating apoptotic signals

5- Tumor-Suppressor Genes (p53)

• The cell is also subjected to many other apoptosis

modifiers including:

1) Various cytokines (e.g., IL-4, IL-2, IL-10),

2) Circulating or membrane-bound molecules which are

capable of triggering specific ligand-receptor

interactions (e.g., Fas ligand/Fas, TNF-α /TNF-RI),

Nitric oxide

6- Other Apoptosis Modifiers

Nitric oxide:

• It is a potent pro-inflammatory molecule with a role in various

pathological conditions, such as joint damage in rheumatoid arthritis

and osteoarthritis, as well as in diseases associated with vascular

dysfunction. It has dual action:

• Pro-apoptotic activity: where it increased the mitochondrial

membrane potential releasing cytochrome c that subsequently

activates caspase-3.

• Anti-apoptotic activity : where induces the S-nitrosylation of

caspases, thereby preventing their activation and promoting

resistance to Fas-mediated apoptosis.

• Difference between apoptosis and necrosis

Cellular

Modification

Apoptosis Necrosis

Cellular role Active Passive

Distribution Dispersed Contiguous

Morphology Decreased

volume of

the cell

Increased

volume of the

cell

Cellular

membrane

Preserved Loss of

integrity

Induction Slow

(hours)

Rapid

(seconds–

minutes)

Cell removal Rapid Slow

Tissue

inflammation

Absent Present

• Angiogenesis is the formation of new blood vessels from pre-

existing ones.

• Vasculogenesis is development of blood vessels from in situ

differentiating endothelial cells.

Angiogenesis

Steps of angiogenesis:

1. Local basement membrane degradation of the parent vessel

2. Locomotion of endothelial cells away from the parent vessel in the

direction of an angiogenic stimulus

3. Elongation of endothelial cells to form a capillary sprout

4. Endothelial cell proliferation in the parent venule and in the capillary sprout

5. Lumen formation

6. Anastomosis of two hollow sprouts to form a capillary loop,

7. Onset of blood flow

8. Production of new basement membrane.

Regulation of angiogenesis:

• Angiogenesis is a complex process regulated by a balance

between angiogenic and antiangiogenic factors

1. Angiogenic factors

• Includes variety of cytokines and growth factors such as:

• Vascular endothelial growth factor (VEGF):

• It is a key regulator of both physiological and pathological

angiogenesis.

• Its biological effect is mediated through 3 receptor tyrosine

kinases; VEGFR-1, VEGFR-2 and VEGFR-3. Also, a soluble

form of VEGFR-1 (sVEGF-R1) had been identified.

2. Anti-angiogenic factors: such as

• Thrombospondin and Pigment epithelium derived growth

factor (PEDF)

• PEDF is the most recently discovered antiangiogenesis factor.

• It can significantly reduce tumor neoangiogenesis and tumor

growth in animal models with HCC and lewis lung carcinoma.

Angiogenesis in physiological and pathological

conditions:

• Physiologically, it is essential for embryonic development,

menstrual cycle, and wound repair.

• Pathologically: Unregulated angiogenesis is seen in

several pathological conditions including: psoriasis,

nephropathy, cancer, retinopathy, rheumatoid arthritis,

obesity, infectious diseases, etc.

• Angiogenesis can be considered as a therapeutic target

in the treatment of some diseases.

Therapeutic angiogenesis:

• Stimulation of angiogenesis can be used in ttt of:

1) Myocardial ischaemia

2) Cerebral ischaemia

3) Peripheral ischaemia (peripheral arterial occlusive

disease)

4) Wound healing and fracture repair

5) Reconstructive surgery: Skin flaps

6) Induction of collateral vessel formation

• Inhibition of angiogenesis can be used in:

1) Inhibition of tumor growth and metastasis.

2) Treatment of ocular neovascularization.

3) Treatment of haemangioma

4) Treatment of rheumatoid arthritis

5) Treatment of atherosclerotic plaque

neovascularization

Targets of angiogenesis inhibitors:

1) Inhibition of exogenous or endogenous angiogenic

growth factors or their receptors.

2) Inhibition of endothelial cell growth and survival.

3) Some inhibitors target the basement membrane and

extracellular matrix.

4) Inhibition of angiogenic signalling pathway.

FDA approved angiogenesis based treatments:

1) Avastin

• It is the first approved angiogenesis based anticancer agent

• It is a recombinant humanized monoclonal antibody directed

against VEGF.

• In combination with 5FU it is significantly effective against

metastatic colon cancer

FDA approved angiogenesis based treatments:

2) Regranex gel

The active ingredient is becaplermin (a recombinant

human platelet-derived growth factor, PDGF)

It is approved by FDA for topical administration in

treatment of diabetic neuropathic foot ulcers.

• Growth factors are polypeptides exert a mitogenic response

on their target cells.

• They affect many different types of cells e.g. blood cells,

nervous system, mesenchymal tissues and epithelial tissues.

• Types: Growth factors act like hormones:

• Endocrine manner: pass in circulation to their target cells.

• Paracrine manner: affect neighboring cells.

• Autocrine manner: affect their secreting cells

Growth factors

• Clinical implication of growth factors:

• They are implicated in pathogenesis, diagnosis and prognosis of

many diseases including:

1. Diabetes mellitus: in certain types there is absence of insulin as

a growth factor and as a hormone that stimulates the secretion of

other growth factors as IGF-I.

2. Cancer aggression.

3. Chronic inflammatory diseases: due to defective control on

angiogenesis through growth factors.

4. Neurogenerative diseases: due to defective nerve growth factor

and pro-apoptotic cytokines.

• Examples of growth factors:

Growth Factor Source Function

Epidermal growth factor

(EGF)

Mouse salivary gland Stimutates growth of many

epidermal and epithelial cells

Erythropoietin Kidney Regulates development of

early erythropoietic cells

Fibroblast growth factors

(FGFs) (at least nine family

members)

Many different cells Promote proliferation of many

cells

lnterleukin (IL-1) Conditioned media Stimulates production of IL-2

by T cells

lnterleukin-2 (IL-2) Conditioned media Stimulates growth of T cells

Nerve growth factor (NGF) Mouse salivary gland Tropic effect on sympathetic

and certain sensory neurons

Platelet-derived growth factor

(PDGF)

Platelets Stimulates growth of

mesenchymal and glial cells

Transforming growth factor

(TGF)

Conditioned media of

transformed or tumor cells

Similar to EGF

Transforming growth factor

(TGF)

Kidney, platelets Exerts both stimulatory and

inhibitory effects on certain

cells.

Thanks