Fundamentals of Cancer Cellular and Molecular

Biology and Radiobiology

The Biology and Genetics of

Cells

Teresa Martins, PhD

Radiobiology – Medical Physics
Portuguese Institute for Oncology at
Coimbra

tmartins@ipocoimbra.min-saude.pt
Regulation of gene expression
 12. Small non-coding RNAs (e.g., miRs)
• More than 400; regulate at least 1/3 of all protein coding genes
• Base-pair w/ specific mRNAs and regulate their stability and translation
miRs form a double-stranded (ds) structure –
sequence complementarity between different
miRs precursors are parts of the miR
synthesised by RNApol II, miRs undergo processing
capped and polyadenylated (cropping) and are exported
to the cytosol

miRs are further

miR is assembled with cleaved by DICER –
Argonaute and other mature miR
proteins – RNA-induced
silencing complex - RISC RISC cleaves and discards
one of the miR strands - ss
ss-miR guides RISC
to specific mRNA
through base-
pairing (7nt, 3’UTR) Less extensive match: Argonaute
does not cleave mRNA;
translation is repressed and
Extensive match: mRNA destabilised.
mRNA cleaved by
Argonaute – removes Shortening of poly-A
poly-A-tail; exposure tail and transfer into P-
to exonucleases bodies – decapped and
degraded
After mRNA cleavage, RISC and the associated miR are released and can
seek out additional mRNA molecules → a single miR can act catalytically to
destroy many complementary mRNAs.

Several features make miRs especially useful regulators of gene

expression:

1. A single miR can regulate a whole set of different mRNAs (hundreds!), as

long as the mRNAs carry a common sequence in their UTRs

2. Regulation by miRs can be combinatorial – when the base pairing between

the miR and mRNA fails to trigger cleavage, additional miRs can bind to
mRNA leading to further reduction in translation

3. miRs occupy relatively little space in the genome when compared with
proteins
 RNA interference (RNAi) is a cell defence mechanism

• Many of the proteins involved in miR regulatory mechanisms also serve defence
functions
• Degradation of foreign RNA molecules, particularly dsRNAs (e.g., viral infections)
• Attraction of a protein complex containing DICER

Cleaves dsRNA into small fragments (23nt)

– small interfering RNA (siRNA)

Bound by Argonaute and other RISC proteins

→ one of the strands is cleaved – ss siRNA Ss siRNA

Exact match

Directs RISC back to complementary viral

dsRNAs → exact match → cleavage by
Argonaute → fast destruction
The RNA interference machinery can selectively silence synthesis of target
RNAs
siRNAs produced by DICER are assembled
with a protein complex that includes
Argonaute – RNA-induced transcriptional
silencing - RITS

ss-siRNAs are used as sequence guides to

bind the complex to complementary RNA
transcripts

RITS attracts proteins that covalently modify nearby

histones or methylate DNA – formation and spread of
heterochromatin

Transcription repression

RNAi-induced heterochromatin formation important in: 1)

cell defence; 2) normal cell processes. E.g., maintenance
of heterochromatin formed around centromeres
Centromeric DNA transcribed in both directions → complementary
RNA transcripts → dsRNA → siRNA
Crucial for accurate chromosome segregation during mitosis
 Protein Regulation

• Biological properties of proteins – determined by interaction with other

molecules, through the formation of weak noncovalent bonds
• Weak or strong interactions
• High specificity
• Molecules that bind to the protein active site (binding-site) – Ligands
• Different regions of the protein surface provide binding-sites for distinct
ligands – regulation of protein activity
• Other parts of the protein can act as a handle to position the protein in the
cell
Living cells contain thousands of
enzymes that catalyse a myriad of
chemical reactions…

Many of these take place

simultaneously in the small cytosol
volume…

Complex web of metabolic

pathways, each composed of
chains of chemical reactions –
cycles, chains, cascades

Elaborate controls are required to

regulate when and how each
reaction occurs
Regulation can occur at various levels:

1. Control of how many molecules of each enzyme is produced through

regulation of gene expression

2. Confinement of sets of enzymes to particular subcellular compartments

enclosed by distinct membranes

3. Control of activity by covalent modification of proteins

4. Control of the rate of protein destruction by targeted proteolysis

5. Adjustment of the reaction rates through a direct, reversible change in

the activity of the enzyme in response to specific small molecules that

the enzyme encounters (most general process).

Enzymes have at least two different binding sites: 1) active site (substrates)
and 2) regulatory site (regulatory molecules)
There is communication between the two sites → Conformational changes
- binding to one site causes a shift from one folded shape to a slightly
different one

Positive Regulation

Negative Regulation
Proteins are regulated by more than the reversible binding of other molecules…
Another method: covalent addition of a smaller molecule to one or more aa side
chains
The most common: phosphorylation – addition of phosphate groups by kinases
Can affect proteins in two ways: 1) activation/deactivation

Two (-) charges → major conformational Phosphatases removes phosphate groups

change through attraction a cluster of → protein returns to its original
(+) charged aa side chains → altered conformation → basal activity restored
ligand-binding → dramatic changes in
activity

2) Phosphorylated side chains can form part of a structure recognised by the

binding sites of other proteins → protein phosphorylation and dephosphorylation
may drive regulated assembly and disassembly of protein complexes
Reversible phosphorylation controls activity, structure and cellular localization
Hundreds of protein kinases in the cell are organized into complex networks of
signalling pathways that help coordinate cell’s activities, drive cell division, and
relay signals into the cell from the microenvironment.

Extracellular signals need to be integrated and amplified (cascades).

Kinases serve as input-output devices in the integration of signals

An important part of the input of signal processing proteins comes from the
control exerted by phosphorylation (kinases) and dephosphorylation
(phosphatases).

But protein regulation can be much more complex… Many proteins are controlled
by multiple covalent modifications – Combinatorial regulation
 Combinatorial Regulatory Code
- set of covalent modifications of a protein -

Central role in the control of the

cell’s response do adverse
circumstances

Through one of four types of molecular additions, p53 can be modified at 20

different sites!
Because an enormous number of different combinations of these 20
modifications are possible, the protein’s behaviour can be altered in a huge
number of ways.
• Each protein’s set of covalent modifications constitutes an important
combinatorial regulatory code
• As specific modifying groups are added/removed, the code causes different
sets of protein behaviours
• Allows cells to respond rapidly and with great versatility to changes in their
condition or environment
 Mechanisms of cell-cell communication
• To make a multicellular organism, the cells must communicate
• Complex intracellular mechanisms are needed to control which signals are
emitted at what time, and to enable the signal-receiving cell to interpret
these signals and use them as a guide to behaviour
• Communication between cells is mediated mainly by extracellular molecules
that may act over long distances, signalling cells far away, or may signal only
to immediate neighbours
• Most cells are both emitters and receptors

Ligand binding activates the receptor

→ activation of intracellular signalling
pathways
Relay chains of molecules
Process the signal and
transmit it to effector
proteins

Altered when the signalling pathway is Different signals

activated and implement the adequate and nature and
response state of receptor
cells → different
effectors
Types of signal molecules: proteins, small peptides, aa, nucleotides, steroids,
retinoids, fatty acid derivatives, dissolved gases (e.g., NO, CO)
↓
Released to the extracellular space by exocytosis, diffusion through the cell’s
membrane, displayed on the external surface of the cell (implies cell-cell
contact). ↓
Transmembrane proteins
Their extracellular domain can also be released (secretion) and act at a distance

Receptors on the target cell are mainly transmembrane proteins (cell-surface

receptors), although there are also intracellular receptors (diffusion)

NO, CO, steroid hormones,

thyroid hormones, retinoids, vit.D

Signal molecule –
IR complexes act
as transcription
factors-
Nuclear Receptor
Family
 Types of Signalling
Signal molecules remain bound the Secreted signal molecules; may
the surface of signalling cell and act as local mediators affecting
only influence cells that contact it only neighbouring cells; fast.

Usually, signalling and target cells

are different. However, cells may
also produce signals that they
themselves respond to
- Autocrine signalling

Secreted signal molecules;

carried far afield to act on
distant cells; entry into the
blood stream; slow.
The speed of the response to an extracellular signal depends on:
1. The mechanism of delivery
2. The nature of the target cell’s response

When the response

only requires changes
in proteins already
present in the cell.
E.g., phosphorylation,
opening if ion-channels

When the response

requires changes in
gene expression
and protein
synthesis
Cells can also communicate directly through gap junctions:
- Narrow, water-filled channels that directly connect the cytoplasm of
adjacent epithelial cells
- Allow the exchange of inorganic ions (e.g., Ca2+) and other small water-soluble
molecules (e.g., cAMP)
- Allow communication to pass in both directions symmetrically
- Their typical effect is to homogenize conditions in communicating cells
A typical cell in a multicellular organism may be exposed to hundreds of different
signal molecules in its environment at a given time…

Signal molecules may be soluble, bound to the extracellular matrix, or bound to

the surface of neighbouring cells.

Signal molecules can be stimulatory or inhibitory.

Signal molecules can act in innumerable different combinations and influence

almost any aspect of cell behaviour.

The receiving cell must response to this myriad of signals

selectively, according to its own specific character –
acquired through progressive specialization during
development (commitment and differentiation).
- differentiation
- division
- specialized functions (e.g., contraction, secretion)
- live or die

The decision to live depends on the correct interpretation of combinations of

survival signals. When deprived of these signals, the cell activates a suicide
programme and kills itself – apoptosis or programmed cell death.
 1st messengers
2nd messengers:
- small intracellular molecules the
relay the signal.
- Generated in large numbers
- Often diffuse away from the
source – signal spreading
- Water-soluble – diffuse in cytosol
(e.g., cAMP, Ca2+)
- Lipid-soluble; diffuse in the plane
of the plasma membrane (e.g.,
DAG)
- Bind and alter conformation and
behaviour of signalling or effector
proteins

Large intracellular signalling

molecules:
- Help relay the signal by generating
2nd messengers or activating the
next signalling or effector proteins
- form a functional network in which
each protein helps to process the
signal
Large intracellular signalling molecules:
Spread the signals influence through the
cell in one or more ways:
1. Simply relay the signal to the next
signalling component in the chain
2. May act as a scaffold bringing together
2 or more signalling proteins – quicker and
more efficient action
3. May transform (transduce) the signal
into a different form – suitable for passing
the signal along, or to stimulate cell
response
4. May amplify the signal – production of
large amounts of 2nd messengers, or
activation of many copies of downstream
signalling proteins:
- Small number of extracellular
molecules elicit large intracellular
response → amplification →
signalling cascade
5. May receive signals from 2 or more signalling pathways and integrate
them before relaying the signal onward

e.g., A and B trigger two different

signalling pathways, each of which
lead to phosphorylation of the same
target Y, but on different sites.
Y is only activated when signals A and
B are simultaneously present
6. May spread the signal from one
signalling pathway to another – creation
of branches to be activated – increase in
the complexity of the response.

7. May anchor one or more signalling

proteins in a pathway to a particular cell
structure where the signalling proteins
are needed.

8. May modulate the activity of other

signalling proteins and thereby regulate
the strength of signalling along the
pathway.
 Cells can adjust their sensitivity to a signal – Adaptation/Desensitization

A strong response makes the machinery reset itself to become less responsive to
the same level of signal; Underlying mechanism: negative feedback machinery
that operates with a short delay.

Inactivation of the receptor: Downstream of receptor:

- Endocytosis and temporary sequestration in - Changes in intracellular signalling
endosomes upon ligand binding proteins
- Endocytosis followed by lysosomal degradation – - Production of inhibitor proteins
receptor down-regulation that blocks signal transduction
- Inactivation by receptor modification
(phosphorylation or methylation)
 The Cell Cycle
Multicellular species: long and complex sequences of cell divisions produce a
complete new organism. Even in the adult body, cell division is still needed to
replace cells that die.

If cell division was stopped, we would die within a few days…

During cell division, two complete copies of the genome and the replicated
chromosomes must be accurately distributed (segregated) to the two daughter
cells.
Cells must also duplicate all the organelles and macromolecules. Otherwise,
daughter cells would get smaller in each cell division… to maintain their size,
dividing cells must coordinate their growth.
 Eucaryotic Cell Cycle is divided in 4 phases:
- The two major phases are defined by two major requirements: accurate
genome duplication and accurate segregation of the information into the two
daughter cells
M phase –<1h; Chromosome
segregation and cell division
Mitosis – nuclear
division; chromosome
segregation

Cytokinesis –
cytoplasmic
S-phase – 10-12h division
Chromosome
duplication
End of S-phase: the DNA molecules in each pair of duplicated chromosomes are
intertwined and held tightly together.

Prophase:
- early mitosis Prometaphase:
- the 2 DNA molecules - Nuclear envelope
gradually disentangled disassembles
- DNA condensation into - Sister chromatids
pairs of rigid and attach to mitotic spindle
compact rods
- Sister Chromatids
Metaphase:
- Sister chromatids attach to opposite poles of the mitotic spindle and eventually align
at the spindle equator

Anaphase:
- Sister chromatids cohesion is destructed and
they are pulled to opposite poles of the spindle

Telophase:
- The spindle is
disassembled and
segregated chromosomes
are packaged into
separate nuclei

Cytokinesis:
- Cleaves the cell in two
Most cells require much more time to grow and double their mass of proteins
and organelles than they require to duplicate their genome and divide…

These events occur during the gap phases: G1 and G2

M phase
Interphase Aprox 1h…
Aprox 23h…

Gap phases:
- More than simple time delays to allow cell growth
- Provide time for the cell to monitor the internal and external environment
- Provide time for preparations to be completed before the cell commits
itself to the major upheavals of the S- and M-phases
Eucaryotic cells have evolved a complex network of regulatory proteins that
governs progression through the cell cycle
↓
Cell Cycle control system

The core of this system is an ordered series of biochemical switches (cell

cycle clock) that initiate main events, including chromosome duplication and
segregation.

There are additional layers of regulation (checkpoints) to enhance the fidelity

of cell division and allow the control system to respond to various extracellular
and intracellular signals .
↓
The control system monitors progression through the cell cycle and delays later
events until earlier events have been completed

The control system also monitors extracellular conditions – highly responsive to

signals from other cells, stimulating cell division when more cells are needed and
blocking it when they are not needed.

The cell cycle control system has a central role in regulating cell numbers in
body tissues.

When the system malfunctions, excessive cell divisions can result in cancer…