International School of Biolelectromagnetics

“Alessandro Chiabrera”

Analysis of cellular functions:

cell cycle, proliferation, apoptosis

Isabelle Lagroye
PIOM Laboratory -Bioelectromagnetics group
Can low-level electromagnetic fields

affect basic cellular functions?

A 20-year-old question
Agents that are able to…

 Alter cell viability, induce cell death

 Affect the cell cycle,
 Decrease cell proliferation,

… are called cytotoxic agents

They have the potential to destroy living cells
Cytotoxicity
all types of agents concerned
 Genotoxic agents : DNA is directly
affected
 Non genotoxic agents : DNA is not
affected or affected indirectly

 Many tools available for the investigation

 New endpoints as the knowledge in cellular
biology increases
The cell is the fondamental unit of live matter
autonomy, proliferation

Membrane
Nucleus
Cytoplasm

DNA
 The central paradigm

Information source
(A, T, C, G) Control all cellular activities
”order the job”

Information messengers
(A, U, C, G) Result from the DNA transcription
Information transferred to the cytoplasm
”transfer the job”

Final effectors
Result from the RNA traduction
”do the job”
Cell cycle : definition
Alternance of
 mitoses (cell division)
 interphases (metabolic activity)

M-phase  

M-phase S- phase
préparation preparation

DNA synthesis
 DNA and cell cycle

DNA
amount in
the nucleus
S-phase cells
=
Proliferating cells

time
 Cell cycle : G1-S and G2-M checkpoints

 They prevent the cell

to enter into critical
phases while damaged

 Those checkpoints are

under the control of cyclin-
dependent kinases
Cell cycle
 The different phases of the cell cycle are determined
based on the DNA amount in the cells

 DNA is stained using Propidium iodide (+ RNase)

 DNA amount is calibrated

Stained cells Solution

Lens
 Detection of the
amount of DNA in
n
single cells using s
is s
io Cell physical
e c e ll
m characteristics
flow cytometry c
t
en ed
s n
o re s ta i
u f
Fl o
Cell cycle
Example: G2/M cell cycle arrest after ionising radiation in
Hela cells

9.5 11 12.5 h
 Proliferation assays
 During S-phase, the cell replicates its
genome.
If labeled DNA precursors are added to the
culture medium, cells actively synthesizing
DNA will incorporate the precursors into their
DNA.
The incorporated precursor can be detected.

Two precursors are used:

 3H-thymidine : detection by counting incorporated
radioactivity

 BromoDesoxyUridine (BrdU) : detection by using

specific antibodies that are specific for BrdU.
 The cells in culture are loaded with BrdU or 3H-thymidine and
incubated for different duration i.e. 2, 4, 8, …etc hours
 A pulse chase is performed (removes non-incorporated
precursors)
 Proliferating cells are detected
Apoptosis or
Programmed cell death
 Major role in physiology
 involved in morphogenesis / development
i.e., interdigital tissues regression

 involved in cell and tissues homeostasis

Proliferation Apoptosis
In 7 years : renewal of
the whole body
 Major role in pathology

 Dysregulation of apoptosis leads to disease

Defective apoptosis Excessive apoptosis

Apoptosis Proliferation

Proliferation Apoptosis

Neurodegenerative or
Cancer auto-immune diseases

 New target in therapy

Induction of apoptosis in apoptosis-resistant cancer cells
 Two types of cell death
NECROSIS
Mitochondrial changes Membrane breakdown

chromatin pattern conserved

normal reversible swelling irreversible swelling disintegration

APOPTOSIS
Mitochondrial structure preserved Intact membrane

Nuclear changes Apoptotic bodies

normal condensation (cell blebbing) fragmentation secondary necrosis
Apoptosis
signalling
Apoptotic parameters
 Numerous tools for apoptosis detection
 Recommended to investigate 2 markers
 Characteristic pattern of morphological, biochemical
and molecular changes
Apoptotic parameters: morphology
Use of fluorescent dye (DAPI, PI, Hoescht 33342, etc)

 Allows the visualisation of chromatin

condensation (microscopy)

EA.hy926 cultures treated with

Poly-Hema and Fas

sub-
 Using flow cytometry G1
and cell cycle peak
Apoptotic parameters: caspase 3
 Family of caspases (14) : Cystenyl aspartic acid proteases
 Cleave numerous substrates (aspartate residue) upon
induction of apoptosis
 Caspase 3: convergent point of the two-described
apoptosis pathways

Extrinsic
pathway

Intrinsic
pathway
 Substrate (Ac-DEVD)
Caspase 3 contained
in the sample
Coloured product

U937 cells incubated with camptothecin (CAM)

Apoptotic parameters:
cell membrane alteration

Normal cell Apoptotic cell

inner leaflet inner leaflet

outer leaflet outer leaflet

no phosphatidylserine phosphatidylserine on the

on the outer leaflet of the outer leaflet of apoptotic cell
normal cells membrane
  Detection of the presence of phosphatidyl-
serines on the cell membrane: Annexin V
 Detection of necrotic cells: Propidium Iodide
Necrotic cells
Annexin V PI PI+

Normal

Apoptotic

Viable cells Apoptotic cells

ANX— / PI— ANX+ / PI—
Apoptotic parameters:
DNA fragmentation - TUNEL
 During apoptosis, strand breaks (”nicks”) are introduced
into the DNA via enzymatic activity (nucleases).

 Identification
of the breaks by
labeled precursors using
enzymatic reactions: terminal
transferase (TdT).

 This sensitive method is called

TUNEL (TdT-mediated dUTP
nick-end labeling).
TUNEL assay in:
Cell culture

Tissus sections
Programmed cell death
 Highly regulated phenomenon

Stimulus Regulation
Survival
Signals Intracellular Signal
mediators integration
PI3K, PKB
Cell damage ?
P53
(cytotoxic agents, Effectors
Bcl2
heat, etc)
Ceramides
cytochromeC Caspases
Cell receptors Nucleases Death
Oncogenes,
(Fas, TNF, etc)
…
…
Proteolysis
DNA degradation

 Alteration at the level of mediators does not

necessarily mean apoptosis induction
Cytotoxicity assays
 Assays that measure plasma membrane damage, leakage
 Assays that measure metabolic activity in cell populations

Decrease of mitochondrial metabolism

 Cytotoxicity assays: membrane integrity
Lactate dehydrogenase (LDH): stable cytoplasmic enzyme
present in all cells, rapidly released into the cell-culture
supernatant when damage occurs to the plasma membrane
 based on plasma-membrane leakage,
 detects the late stage of cell death,
 no metabolic labelling is necessary,
 safe and convenient alternative to radioactive methods
Cytotoxicity assays: metabolic activity
 Tetrazolium salts (MTT, XTT, and WST-1) are cleaved to
formazan by the ”succinate tetrazolium reductase” system
(respiratory chain of mitochondria), and is functional only in
metabolically active cells.

 The generated dye can thus be quantitated

(spectrophotometer) by measuring the absorbance at
appropriate wavelengths.

Measures the metabolic activity

Used for studying viability and cell proliferation
Widely used (commercial kits)
 Example using WST-1 dye

MTT must be solubilized prior to photometric detection. XTT produces a

water-soluble cleavage product, but is less stable than WST-1. Moreover,
WST-1 has a wider linear range and exhibits accelerated color
development compared to XTT
 Can low-level electromagnetic fields
affect basic cellular functions?
A 20-year-old question

 Modern and sensitive techniques are available

to answer the question

 It is of importance to distinguish between

cytotoxicity in normal and cancer cells

 Integrative aspects of cell functions: cell cycle,

proliferation, apoptosis should not be neglected