Optimisation

of

Biochemical

Condition

and

Substrates

In

Vitro

for

Tissue

Engineering

of

Ligament

In

this

work

we

analysed

the

effect

of

growth

factors

on

in

vitro

cell

proliferation

and

collagens

synthesis

by

fibroblasts

cultured

for

h

on

different

substrates

-LRB-

silicon

sheet

with

or

without

gelatin

and

glass

as

control

surface

-RRB-

for

ligament

tissue

engineering

A

human

fibroblast

cell

line

-LRB-

-RRB-

was

used

The

synthesis

of

type

I

and

type

III

collagens

were

evaluated

qualitatively

and

quantitatively

by

RT-PCR

and

confocal

microscopy

respectively

Cell

proliferation

was

evaluated

by

two

methods

-LRB-

-RRB-

MTT

assay

-LRB-

-RRB-

cell

cycle

analysis

It

was

found

that

PDGF-AB

stimulate

the

proliferation

of

fibroblast

cultured

on

gelatin

coated

silicon

sheet

in

dose

dependant

manner

with

a

maximum

effect

at

ng

ml

The

exogenous

induced

the

expression

of

type

I

and

type

III

collagens

in

a

dose

and

substrate-dependant

manner

We

deduce

from

this

work

that

biochemical

conditions

and

substrates

have

an

important

impact

for

optimisation

of

the

tissue

neo

synthesis

INTRODUCTION

Tissue

engineering

is

a

multidisciplinary

field

at

the

crossroads

of

biochemistry

mechanics

engineering

and

materials

science

to

develop

substitutes

for

replacing

injured

or

diseased

tissues

For

all

applications

it

is

necessary

to

take

into

account

of

several

points

cell

source

growth

factors

mechanical

forces

substrates

and

the

behaviour

of

the

cells

on

the

substrate

Substrates

or

scaffolds

are

defined

as

the

matrix

that

provides

a

suitable

environment

for

cell

proliferation

and

differentiation

Some

scaffolds

have

been

prepared

from

natural

and

synthetic

materials

such

as

collagen

poly

-LRB-

glycolic

acid

-RRB-

-LRB-

PGA

-RRB-

and

copolymers

of

l-lactic

acid

and

є-caprolactone

It

is

necessary

to

improve

the

physical

and

chemical

nature

of

scaffold

for

the

superior

induction

of

tissue

regeneration

However

the

chemical

composition

of

the

scaffold

can

have

great

influence

on

its

biocompatibility

The

source

solubility

degree

of

cross

linking

physical

form

of

the

protein

coating

substrates

and

the

substrates

itself

can

influence

the

cell

functions

like

proliferation

and

metabolic

activity

Cells

and

materials

are

two

essential

components

in

ligament

tissue

engineering

Materials

could

interfere

with

cells

adhesion

proliferation

and

differentiation

Currently

there

are

two

common

approaches

to

modify

the

materials

to

improve

their

biocompatibility

coating

with

biocompatible

materials

or

surface

modification

As

nearly

all

interaction

between

cells

and

artificial

surfaces

are

mediated

by

a

layer

of

absorbed

protein

many

proteins

have

been

coated

on

the

surface

of

target

materials

to

promote

adhesion

-LRB-

i.e.

fibrinogen

collagen

hyaluronan

-RRB-

Protein

coated

substrates

are

provided

as

a

two

dimensional

substrates

Moreover

it

has

been

shown

that

fibroblast

attachment

morphology

proliferation

and

collagen

synthesis

varied

from

different

culture

substrates

and

the

type

of

the

fibroblast

obtained

from

different

organs/tissue

An

appropriate

scaffold

for

ligament

regeneration

must

be

able

to

provide

mechanical

strength

to

initially

withstand

the

in

vivo

force

and

degrade

safely

at

an

appropriate

rate

in

vivo

In

vitro

studies

a

planar

artificial

substrate

-LRB-

i.e.

silicon

membrane

-RRB-

is

usually

used

for

growing

cells

and

subjected

them

to

mechanical

loading

Furnishing

scaffolds

with

biochemical

factors

are

also

promising

Many

biochemical

factors

including

growth

factors

could

promote

tissue

regeneration

and/or

healing

processes

Growth

factors

are

known

to

play

an

important

role

in

the

healing

response

of

connective

tissues

Several

reports

indicate

the

significance

of

growth

factors

in

the

intrinsic

healing

process

of

injured

ligaments

Many

growth

factors

affect

the

tendon

and

ligament

healing

process

after

injury

by

stimulating

extra-cellular

matrix

-LRB-

ECM

-RRB-

synthesis

and

cell

proliferation

It

has

been

shown

that

exogenous

Transforming

Growth

Factors

β1

-LRB-

-RRB-

increased

the

collagen

synthesis

of

human

anterior

cruciate

ligament

cells

and

human

ligamentum

flavum

Platelet

Derived

Growth

Factor-AB

-LRB-

PDGF-AB

-RRB-

one

of

the

major

serum

growth

factor

is

known

to

enhance

cell

proliferation

and

healing

responses

as

well

as

to

increase

rupture

force

stiffness

and

breaking

energy

of

the

tissue

The

definition

of

the

optimal

condition

of

culture

such

as

coating

of

the

substrate

synergistic

effect

of

the

substrate

and

growth

factors

are

required

for

ligament

tissue

engineering

In

order

to

define

the

optimal

concentration

of

growth

factors

and

the

evolution

of

cells

culture

on

silicon

membrane

which

will

be

subjected

to

cyclic

stretch

for

later

mechanical

study

the

objective

of

the

present

work

was

to

investigate

the

influences

of

individual

growth

factors

and

substrates

on

the

cell

proliferation

and

ECM

synthesis

In

this

work

we

studied

the

cell

viability

proliferation

collagen

production

of

fibroblast

seeded

on

the

different

substrates

and

the

effect

of

PDGF-AB

on

the

cell

proliferation

and

TGF-β

on

the

ECM

synthesis

to

allow

the

optimisation

of

culture

conditions

which

could

be

used

for

in

vitro

reconstruction

of

ligament/tendon

tissues

MATERIALS

AND

METHODS

Cells

and

Culture

Conditions

Human

skin

fibroblast

cell

line

-LRB-

-RRB-

was

purchased

from

American

Type

Culture

Collection

-LRB-

ATCC

No.

Manassas

VA

-RRB-

The

cells

were

placed

in

plastic

culture

flask

with

Dulbecco

's

modified

Eagle

's

medium/Nutrient

-LRB-

GibcoBRL

Life

Technologie

France

-RRB-

containing

decomplemented

foetal

bovine

serum

-LRB-

FBS

-RRB-

supplemented

with

U/ml

penicillin

μg/ml

streptomycin

mM

l-glutamine

μg/ml

fungizone

non-essential

amino

acids

-LRB-

GibcoBRL

Life

Technologie

France

-RRB-

μg/ml

l-ascorbic

acid

g/l

-LRB-

Sigma

France

-RRB-

and

were

incubated

at

°C

-LSB-

air

-LRB-

-RRB-

and

-LRB-

-RRB-

-RSB-

The

medium

was

changed

every

days

At

confluence

cells

were

detached

from

the

culture

flask

with

trypsin

EDTA

mixture

-LRB-

Sigma

France

-RRB-

for

min

at

°C

and

then

resuspended

in

complete

medium

until

confluence

Substrate

Surface

Treatment

Transparent

elastic

silicon

sheet

-LRB-

thickness

cm

-RRB-

-LRB-

Specialty

manufacturing

INC

USA

-RRB-

was

punched

out

into

round

sheets

-LRB-

diameter

cm

-RRB-

Glass

slips

-LRB-

diameter

cm

-RRB-

-LRB-

Polylabo

France

-RRB-

and

silicon

sheets

were

sterilised

by

overnight

immersion

in

ethanol

aqueous

solution

air

dried

then

placed

in

a

multiwell

culture

plate

and

sterilised

by

min

UV

irradiation

Gelatin

solutions

-LRB-

w/v

in

distilled

water

-RRB-

-LRB-

Sigma

France

-RRB-

were

sterilised

by

filtration

before

use

Cells

were

seeded

into

wells

culture

plates

at

a

density

of

on

different

surface

glass

slips

-LRB-

control

surface

-RRB-

silicon

sheet

coated

or

not

with

gelatin

for

h

at

°C

to

allow

cell

attachment

Biochemical

Stimuli

The

concentrations

of

growth

factors

used

in

this

study

included

those

noted

to

be

effective

in

previous

studies

The

concentration

range

for

TGF-β

was

ng

ml

and

from

to

ng

ml

for

PDGF-AB

In

our

study

five

concentrations

of

-LRB-

ng

ml

-RRB-

and

four

concentrations

for

PDGF-AB

-LRB-

and

ng

ml

-RRB-

were

used

These

concentrations

were

added

to

the

cultures

in

order

to

determine

their

effects

on

the

cell

proliferation

for

PDGF-AB

and

collagen

production

for

Cell

proliferation

and

collagen

synthesis

were

determined

h

after

the

addition

of

the

growth

factors

Culture

medium

containing

FBS

without

growth

factors

was

used

as

control

Fibroblast

Proliferation

Experiments

MTT

Assay

After

h

PDGF-AB

treatment

MTT

-LSB-

4,5-Dimethylthiazol-2,5-diphenyltetrazolium

bromide

blue

Thiazolyl

-RSB-

-LRB-

Sigma

France

-RRB-

was

prepared

in

PBS

at

a

final

concentration

of

mg/ml

Then

μl

of

the

solution

were

added

to

each

well

containing

μl

of

DMEM

without

red

phenol

The

cells

were

incubated

for

h

at

°C

to

allow

the

yellow

dye

to

be

transformed

into

blue

formazan

crystals

by

the

mitochondrial

dehydrogenases

The

insoluble

product

was

dissolved

by

addition

of

μl

of

acidified

isopropanol

-LRB-

N

HCl

in

isopropanol

-RRB-

and

thoroughly

mixed

to

dissolve

the

dark

blue

crystals

The

supernatants

were

removed

centrifuged

and

read

within

min

by

using

a

Beckman

spectrophotometer

-LRB-

USA

-RRB-

with

a

wavelength

of

and

nm

was

used

as

reference

wavelength

The

optical

density

difference

OD

nm

nm

was

estimated

Cell

Cycle

Analysis

After

h

PDGF-AB

treatment

cells

were

trypsinisation

then

collected

by

centrifugation

-LRB-

-RRB-

for

min

Cells

pellets

were

washed

twice

with

ml

of

PBS

μl

of

the

washed

cell

suspension

-LRB-

cells

-RRB-

were

stained

according

the

kit

instruction

-LRB-

Coulter

DNA

Prep

Reagents

kit

Beckman-Coulter

Miami

USA

-RRB-

in

order

to

analyse

the

cell

cycle

by

flow

cytometry

-LRB-

EPICS

XL

Coulter

Beckman-Coulter

-RRB-

Primary

histograms

were

generated

by

gating

the

cells

according

to

the

surface

and

the

peak

of

the

fluorescence

signal

in

order

to

avoid

taking

doublets

into

account

These

histograms

were

then

submitted

to

computerised

calculations

of

the

cell

cycle

by

using

polynomial

analysis

derived

from

the

Dean

and

Jett

model

Multi

Cycle

AV

-LRB-

Phoenix

Flow

System

San

Diego

USA

-RRB-

For

each

testing

condition

the

percentage

of

cells

in

each

phase

was

determined

DNA

content

was

analysed

in

at

least

nuclei

in

each

sample

RNA

Isolation

and

RT-PCR

Total

cellular

RNA

were

isolated

from

fibroblasts

after

dissolution

in

TRIzol

reagent

-LRB-

Invitrogen

France

-RRB-

according

to

the

manufacturer

's

instructions

RNA

were

extracted

using

chloroform

then

samples

were

centrifuged

-LRB-

-RRB-

for

min

at

°C

Isopropanol

was

added

to

precipitate

the

RNA

and

the

samples

were

centrifuged

for

min

under

the

same

conditions

The

RNA

pellet

was

washed

by

addition

of

ml

of

ethanol

and

centrifuged

at

for

min

The

pellet

was

resuspended

in

μl

DEPC-treated

water

The

concentrations

of

RNA

were

determined

spectrophotometrically

at

nm

and

the

purity

of

RNA

were

controlled

by

the

ratio

nm

The

first

strand

cDNA

was

prepared

from

the

RNA

with

reverse

transcriptase

-LRB-

kit

ThermoScript

RT-PCR

System

-RRB-

-LRB-

BioRad

USA

-RRB-

The

cDNA

was

amplified

by

polymerase

chain

reaction

-LRB-

Platinum

Taq

DNA

Polymerase

-RRB-

-LRB-

Invitrogen

France

-RRB-

using

the

primers

shown

in

Table

Gel

electrophoresis

of

RT-PCR

products

was

performed

in

a

agarose

-LRB-

Sigma

France

-RRB-

run

in

Tris

boric

acid

EDTA

-LRB-

TBE

-RRB-

buffer

-LRB-

Bio-Rad

USA

-RRB-

The

gel

was

stained

in

a

μg/ml

ethidium

bromide

solution

for

min

and

the

images

were

obtained

using

a

Bio-Rad

Molecular

Imaging

System

Bands

intensities

were

evaluated

against

background

quantified

by

Quantity

One

Image

software

-LRB-

Bio-Rad

USA

-RRB-

and

normalized

to

β-actin

TABLE

Prime

sequence

for

RT-PCR

MRNAPrimer

sequenceProduct

size

-LRB-

bp

-RRB-

Type

I

TCC

CCA

GCC

ACA

AAG

AGT

CTA

GTG

ATT

GGT

GGG

ATG

TCT

TCG

Type

III

CTG

CCA

TCC

TGA

ACT

CAA

GAG

CCA

TCC

TCC

AGA

ACT

GTG

TAG

ATC

TGG

CAC

CAC

ACC

TTC

TAC

AAT

GAG

CTG

CGT

CAT

ACT

CCT

GCT

TGC

TGA

TCC

ACA

TCT

Immuonofluorescence

Labelling

After

incubation

with

the

cells

were

rinsed

with

PBS

The

cells

were

incubated

for

min

with

anti-collagen

I

-LRB-

dilution

-RRB-

or

anti-collagen

III

-LRB-

dilution

-RRB-

rabbit

IgG

polyclonal

-LRB-

Calbiochem

France

-RRB-

in

DMEM

without

red

phenol

-LRB-

GibcoBRL

Life

Technologie

France

-RRB-

containing

-LRB-

w/v

-RRB-

BSA

The

cells

were

then

rinsed

thoroughly

in

DMEM

without

red

phenol

and

incubated

with

the

second

antibody

Alexa

conjugated

goat

anti-rabbit

IgG

-LRB-

λex

nm

λem

nm

-RRB-

-LRB-

dilution

-RRB-

for

min

in

the

dark

After

washing

with

DMEM

without

red

phenol

the

cells

were

fixed

with

para-formaldehyde

-LRB-

Sigma

France

-RRB-

in

PBS

for

min

and

rinsed

in

PBS

The

cells

were

observed

under

a

fluorescence

confocal

microscope

-LRB-

micro

systems

Germany

-RRB-

Statistical

Analysis

Differences

in

cell

proliferation

and

collagen

synthesis

were

analysed

by

one-way

ANOVA

analysis

to

evaluate

differences

between

groups

with

PLSD

Fisher

correction

-LRB-

Statview

IV

Abacus

Concepts

Inc

Berkley

CA

USA

-RRB-

A

p

value

less

than

was

considered

statistically

significant

All

data

were

expressed

as

the

mean

standard

deviation

-LRB-

SD

-RRB-

RESULTS

All

the

works

were

realized

on

three

types

of

substrates

-LRB-

silicon

sheet

silicon

sheet

with

gelatin

coating

as

testing

surfaces

and

glass

surface

used

as

a

control

-RRB-

Effect

of

PDGF-AB

on

Cell

Proliferation

We

studied

the

effect

of

PDGF-AB

on

fibroblast

proliferation

according

to

substrates

and

PDGF

concentrations

by

two

different

and

complementary

methods

MTT

Assays

The

results

of

the

MTT

assay

are

summarized

in

Fig.

These

results

were

obtained

from

fibroblast

cultured

on

different

substrates

at

day

in

the

presence

or

absence

of

four

different

concentrations

of

PDGF-AB

On

the

glass

and

gelatin

coated

silicon

sheet

cultures

PDGF-AB

induced

a

dose-dependent

increase

in

the

value

of

On

glass

culture

the

significant

increase

was

observed

in

the

range

of

ng

ml

PDGF-AB

-LRB-

p

-RRB-

On

the

gelatin

coated

silicon

sheet

culture

the

significant

increase

was

observed

in

the

range

of

ng

ml

PDGF-AB

-LRB-

p

-RRB-

On

the

silicon

sheet

culture

the

values

of

decrease

when

the

concentration

of

PDGF-AB

was

above

ng

ml

FIGURE

Dose

dependent

effect

of

PDGF-AB

on

cell

proliferation

by

MTT

assays

Cells

were

treated

with

different

concentrations

of

PDGF-AB

for

h

The

metabolic

activity

of

fibroblast

seeded

on

glass

silicon

sheet

with

or

without

gelatin

was

measured

by

MTT

assays

p

versus

control

glass

p

versus

control

silicon

sheet

p

versus

control

gelatin

coated

silicon

sheet

n

Nevertheless

we

observed

a

loss

of

cells

on

silicon

sheet

at

ng

ml

PDGF-AB

Cell

Cycle

Analysis

To

confirm

the

MTT

results

we

also

studied

the

effect

of

PDGF-AB

on

cell

cycle

-LRB-

Figs.

and

-RRB-

Cell

cycle

analysis

was

performed

on

day

of

the

culture

It

was

observed

in

the

three

types

of

substrates

that

PDGF-AB

induced

a

concentration-dependant

decrease

in

the

percentage

of

cells

in

phase

and

an

increase

in

S

and

phase

FIGURE

Cell

cycle

analysis

by

flow

cytometry

Representative

cell

cycle

histogram

of

the

cells

cultured

on

glass

-LRB-

a

-RRB-

gelatin

coated

silicon

sheet

-LRB-

b

-RRB-

After

days

of

treatment

with

PDGF-AB

-LRB-

ng

ml

-RRB-

cells

were

recovered

and

DNA

staining

with

propidium

iodide

-LRB-

IP

-RRB-

was

performed

Ten

thousand

cells

were

analysed

for

each

sample

n

FIGURE

Effect

of

different

concentrations

of

PDGF-AB

on

fibroblast

cell

cycle

regulation

Cells

cultured

on

glass

-LRB-

a

-RRB-

silicon

sheet

-LRB-

b

-RRB-

gelatin

coated

silicon

sheet

-LRB-

c

-RRB-

Cells

were

subjected

to

different

concentrations

of

PDGF-AB

-LRB-

ng

ml

-RRB-

The

percentage

of

cells

in

each

phase

of

the

cell

cycle

was

analysed

by

flow

cytometry

Results

are

represented

as

mean

SD

of

three

independent

experiments

for

each

substrate

p

versus

control

n

On

gelatin

coated

silicon

sheet

the

percentage

of

cells

in

S-phase

was

higher

than

that

without

gelatin

or

glass

-LRB-

Fig.

-RRB-

Moreover

cells

exposed

to

ng

ml

of

PDGF-AB

had

the

highest

percentage

of

cells

in

phase

-LRB-

p

-RRB-

Effect

of

on

the

Expression

of

Collagens

Effect

of

on

Type

I

and

Type

III

Collagens

mRNA

Expressions

The

gene

expressions

of

collagens

in

cells

cultured

on

different

substrates

were

measured

by

RT-PCR

RT-PCR

analysis

of

type

I

and

type

III

collagen

were

performed

at

day

in

the

presence

or

absence

of

five

different

concentrations

of

The

RT-PCR

results

on

human

fibroblasts

seeded

on

the

three

substrates

are

shown

in

Fig.

The

quantitative

results

concerning

type

I

and

type

III

collagen

are

summarised

in

Fig.

FIGURE

Effect

of

different

concentrations

of

on

the

mRNA

expression

of

type

I

and

type

III

collagen

Representative

RT-PCR

results

of

human

fibroblasts

seeded

on

glass

-LRB-

a

-RRB-

silicon

sheet

-LRB-

b

-RRB-

gelatin

coated

silicon

sheet

-LRB-

c

-RRB-

The

OD

of

each

band

was

normalised

to

the

values

of

housekeeping

gene

β-actin

The

cells

were

cultured

on

different

substrates

in

the

presence

of

different

concentrations

of

-LRB-

ng

ml

-RRB-

for

days.FIGURE

Quantitative

measurement

of

the

RT-PCR

results

a

type

I

collagen

b

type

III

collagen

Results

are

expressed

as

mean

SD

of

a

representative

of

three

different

experiments

carried

out

in

duplicate

Black

bars

represent

cell

culture

on

glass

bright

grey

bars

represent

cell

culture

on

silicon

sheet

dark

grey

bars

represent

cell

culture

on

gelatin

coated

silicon

sheet

p

versus

control

glass

p

versus

control

silicon

sheet

p

versus

control

gelatin

coated

silicon

sheet

n

The

mRNA

expression

of

collagen

type

I

is

shown

in

Fig.

On

the

glass

culture

and

gelatin

coated

silicon

sheet

cultures

there

was

no

significant

difference

in

the

mRNA

expression

of

collagen

type

I

between

the

treated

group

and

the

control

group

Otherwise

on

the

silicon

sheet

culture

the

mRNA

expression

of

collagen

type

I

increased

significantly

in

the

presence

of

ng

ml

The

mRNA

expression

of

collagen

type

III

is

shown

in

Fig.

On

the

glass

culture

we

can

see

a

cyclic

effect

of

on

the

expression

of

collagen

type

III

and

it

reached

a

maximum

response

at

ng

ml

TGF-β

Also

compared

to

control

group

the

mRNA

expression

of

collagen

type

III

was

decreased

significantly

at

a

concentration

of

and

ng

ml

-LRB-

p

-RRB-

On

the

gelatin

coated

silicon

sheet

cultures

the

significant

increase

in

the

mRNA

expression

of

collagen

type

III

was

observed

by

the

addition

of

and

ng

ml

-LRB-

p

-RRB-

On

the

silicon

sheet

culture

the

mRNA

expression

of

collagen

type

III

decrease

when

the

concentration

of

above

ng

ml

Qualitative

Expression

of

Type

I

and

Type

III

Collagens

In

parallel

of

the

RT-PCR

experiments

we

carried

out

some

experiments

by

confocal

microscopy

to

visualise

the

expression

of

collagen

The

protein

expression

of

collagen

type

I

and

type

III

of

the

cells

cultured

on

glass

were

revealed

by

indirect

immunofluorescence

staining

As

shown

in

Fig.

collagen

type

I

occurred

as

spots

dispersed

around

the

cells

and

collagen

type

III

was

organised

as

network-like

fibre

There

is

no

difference

in

the

expression

of

type

I

collagen

with

the

different

concentration

of

In

contrast

the

expression

of

type

III

collagen

was

increased

after

the

addition

of

The

same

expression

model

of

collagens

was

observed

on

the

other

substrates

-LRB-

data

not

shown

-RRB-

FIGURE

Immunofluorescence

staining

of

collagen

in

monolayer

cultures

of

fibroblast

on

glass

visualised

by

confocal

microscopy

-LRB-

A

-RRB-

Type

I

collagen

is

identified

on

the

day

of

culture

by

use

of

anti

collagen

type

I

antibodies

Only

spots

of

this

type

of

collagen

were

secreted

by

the

cells

-LRB-

arrowhead

-RRB-

-LRB-

B

-RRB-

Type

III

collagen

is

identified

by

use

of

anti

collagen

type

III

antibodies

It

appears

like

network

in

the

cell

-LRB-

arrow

-RRB-

-LRB-

objectif

Leica

Germany

-RRB-

Bar

μm

-LRB-

a

-RRB-

Control

cell

-LRB-

DMEM

FBS

-RRB-

-LRB-

b

-RRB-

Cell

treated

with

ng

ml

-LRB-

c

-RRB-

Cell

treated

with

ng

ml

DISCUSSION

As

cell

proliferation

and

matrix

synthesis

are

important

parameters

in

tissue

engineering

the

aim

of

this

study

was

to

optimise

the

in

vitro

culture

conditions

on

ligament/tendon

tissues

for

future

experiments

in

bioreactors

The

effect

of

PDGF-AB

and

on

the

cell

proliferation

and

matrix

synthesis

of

fibroblast

cultured

on

three

different

types

of

substrates

-LRB-

silicon

sheet

with

or

without

gelatin

glass

-RRB-

for

days

were

investigated

in

this

study

For

the

tissue

regeneration

it

is

better

to

use

three-dimensional

scaffolds

to

investigate

the

effects

of

the

properties

of

the

material

on

cell

behaviour

However

there

are

many

parameters

of

the

scaffolds

to

be

considered

such

as

porosity

pore

size

pore

interconnectivity

surface

property

topography

and

chemical

composition

Therefore

in

this

study

two-dimensional

substrates

were

used

to

investigate

the

influence

of

substrate

surface

property

on

the

proliferation

and

ECM

synthesis

of

fibroblast

Firstly

we

used

PDGF-AB

which

was

widely

used

as

a

mitogenic

growth

factor

and

as

a

chemotactic

agent

for

cells

such

as

fibroblasts

Our

experiments

demonstrated

that

cell

proliferation

was

enhanced

by

PDGF-AB

This

result

was

consistent

with

those

of

the

previous

studies

on

lung

fibroblast

canine

anterior

cruciate

ligament

cell

human

periodontal

ligament

-LRB-

PDL

-RRB-

and

human

anterior

cruciate

ligament

cell

-LRB-

ACL

-RRB-

PDGF

increased

the

number

of

fibroblasts

available

to

enter

the

cell

replication

cycle

by

stimulating

termination

of

the

phase

of

the

cell

cycle

The

results

obtained

in

this

study

are

similar

to

those

reported

by

and

which

showed

that

PDGF

would

be

an

effective

growth

factor

that

stimulates

resting

cells

to

advance

from

to

of

the

cell

cycle

Although

the

addition

of

PDGF-AB

resulted

in

significant

increase

in

cell

proliferation

on

all

the

substrates

the

highest

increase

was

found

on

gelatin

coated

silicon

sheet

culture

This

result

may

be

explained

by

the

physical

properties

of

the

substrate

surface

High

cell

proliferation

was

observed

for

the

substrates

with

contact

angles

of

as

well

as

on

protein

coated

culture

plates

such

as

gelatin

Park

et

al.

have

demonstrated

that

mescenchymal

stem

cells

-LRB-

MSCs

-RRB-

cultured

on

collagen

I

or

gelatin

coated

membranes

expressed

similar

levels

of

smooth

muscles

-LRB-

SM

-RRB-

markers

Hori

et

al.

reported

that

the

morphology

and

the

proliferation

of

MSCs

were

influenced

by

the

type

of

substrate

and

culture

medium

The

use

of

gelatin

coated

silicon

sheet

as

a

substrate

allows

to

enhance

the

cell

adhesion

the

differentiation

and

the

proliferation

In

addition

the

higher

response

was

found

to

ng

ml

PDGF

on

gelatin

coated

silicon

sheet

culture

but

not

to

the

maximal

dose

as

ng

ml

PDGF-AB

This

finding

indicates

that

it

exist

a

dose-dependant

effect

of

PDGF-AB

on

cell

proliferation

in

this

culture

model

and

ng

ml

PDGF

might

be

the

optimal

concentration

Indeed

our

result

was

consisting

with

the

previous

experiment

which

demonstrated

that

ng

ml

of

PDGF

promoted

fibroblast

attachment

to

collagen

coated

plates

and

proliferation

Secondly

we

investigated

the

effect

of

on

collagen

synthesis

is

the

prototype

of

a

large

family

of

growth

factors

regulating

cell

growth

development

differentiation

immune

system

and

tissue

repair

On

the

one

hand

mostly

inhibited

the

cell

growth

both

in

vivo

and

in

vitro

It

could

arrests

cell

cycle

progression

at

a

point

in

late

phase

Furthermore

stimulatory

effects

of

PDGF-AB

have

been

demonstrated

to

be

blocked

by

the

presence

of

On

the

other

hand

it

has

been

shown

that

TGF-β

regulates

production

of

the

ECM

including

collagen

fibronectin

and

tenascin

Therefore

in

the

second

step

of

our

works

we

study

the

effect

of

on

the

ECM

synthesis

Our

data

showed

that

the

exogenous

influenced

the

expression

of

type

I

and

type

III

collagen

in

a

substrate

dependant

manner

In

our

study

the

mRNA

expression

of

collagen

type

III

was

higher

than

that

of

collagen

type

I

when

the

cells

were

cultured

on

glass

and

gelatin

coated

silicon

sheet

for

h

During

the

early

phases

of

ligament

healing

and

remodelling

more

type

III

collagen

is

produced

than

type

I

collagen

Therefore

the

greater

upregulation

of

type

III

collagen

as

compared

to

type

I

collagen

is

a

positive

indicator

that

the

cells

are

secreting

signals

for

the

healing

and

remodelling

process

However

on

glass

we

observed

the

highest

expression

of

type

III

collagen

mRNA

in

the

control

group

In

the

present

work

we

used

culture

medium

containing

FBS

Although

we

did

not

add

any

in

the

medium

it

seemed

that

this

increase

was

due

to

the

presence

of

growth

factors

in

the

serum

and

which

normally

stimulate

protein

synthesis

and

other

different

biological

processes

Furthermore

we

found

that

effected

the

expression

of

type

I

and

type

III

collagen

synthesis

by

fibroblasts

with

a

dose

dependant

manner

The

expression

of

type

III

collagen

mRNA

on

gelatin

coated

silicon

sheet

was

significantly

increased

at

and

ng

ml

The

results

of

this

study

are

consistent

with

the

previous

work

which

demonstrated

that

ng

ml

of

may

promote

fibroblast

attachment

In

cell

culture

has

been

demonstrated

to

have

a

dose-dependent

effect

on

collagen

production

In

summary

our

results

demonstrated

that

PDGF-AB

promoted

the

cell

proliferation

which

is

a

basic

step

in

the

process

of

ligament

repair

Our

data

also

suggest

that

is

an

important

cytokine

during

the

early

phases

of

ligament

healing

process

for

its

role

in

the

promotion

of

extra

cellular

matrix

formation

These

results

allowed

us

to

determine

the

main

biochemical

conditions

of

in

vitro

construction

of

ligament/tendon

tissues

Our

results

implied

that

it

is

better

to

use

protein-coating

substrate

to

improve

the

proliferation

and

the

synthesis

of

collagen

for

later

mechanical

stretching

study

Further

studies

are

needed

to

determine

the

combination

of

these

two

growth

factors

and

their

effect

on

the

biological

processes

O O O O O B O O O O O O O O O O O O O O O O B I O O O B I O B I O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O

cell proliferation
collagens synthesis
fibroblasts
growth factors
substrates


Subtypes of oligodendroglioma defined by 1p ,19 q deletions , differ in the proportion of apoptotic cells but not in replication-licensed non-proliferating cells Oligodendrogliomas may be divided into those with deletion of chromosomes 1p and 19q -LRB- Del + -RRB- , and those without -LRB- Del − -RRB- . Del + tumours show better survival and chemoresponsiveness but the reason for this difference is unknown . We have investigated whether these subgroups differ in -LRB- a -RRB- apoptotic index , -LRB- b -RRB- the proportion of cells licensed for DNA replication but not in-cycle , and -LRB- c -RRB- the relative length of G1-phase . Fluorescence in situ hybridisation with probes to 1p and 19q was used to determine the deletion status of 54 oligodendrogliomas , including WHO grades II and III . The apoptotic index was determined using counts of apoptotic bodies . Replication-licensed non-proliferating cells were determined from the Mcm2 minus Ki67 labelling index , whilst the geminin to Ki67 ratio was used as a measure of the relative length of G1 . Del + oligodendrogliomas showed a higher apoptotic index than Del − tumours -LRB- P = 0.037 -RRB- ; this was not accounted for by differences in tumour grade or in proliferation . There were no differences in the Mcm2 − Ki67 index or in the geminin/Ki67 ratio between the subgroups , but grade III tumours showed a higher proportion of licensed non-proliferating cells than grade II tumours -LRB- P = 0.001 -RRB- . An increased susceptibility to apoptosis in oligodendrogliomas with 1p ± 19q deletion may be important in their improved clinical outcome compared to Del − tumours . Introduction Oligodendrogliomas are tumours composed of cells that morphologically resemble oligodendrocytes and that range in spectrum from well differentiated to anaplastic tumours . The current WHO classification of CNS tumours recognises two histological grades , WHO grades II and III , and grading is a significant predictor of survival -LSB- 23 -RSB- . Oligodendrogliomas have generally been considered to have a better prognosis than the corresponding grades of diffuse astrocytoma , and recent data continues to support this -LSB- 21 -RSB- . However , oligodendrogliomas , like astrocytomas , are diffusely infiltrating tumours , precluding complete surgical resection , and they may show malignant progression over time . There is therefore a role for adjuvant radiotherapy and/or chemotherapy , particularly for anaplastic tumours . Approximately two-thirds of oligodendrogliomas show allelic losses from chromosomes 1p and 19q -LSB- 24 , 25 -RSB- , a genetic profile that appears to be associated with clinical outcome . Some anaplastic oligodendrogliomas respond to combination chemotherapy -LSB- 2 , 3 -RSB- , and better progression free survival and chemo-responsiveness appear to be linked to the presence of these deletions -LSB- 4 , 34 -RSB- . Results from two recent large-scale trials provide support for the view that the presence of 1p 19q deletion identifies a subgroup with better clinical outcome -LSB- 1 , 41 -RSB- . 1p 19q deletion may also identify better prognosis in low-grade oligodendrogliomas -LSB- 13 -RSB- . There may be some histopathological differences between the two genetic subgroups . Classical oligodendroglioma features , such as perinuclear haloes -LRB- in > 50 % of cells -RRB- , uniform rounded nuclei , chickenwire vascular pattern and calcification , are associated with 1p 19q loss -LSB- 14 , 27 , 42 -RSB- , suggesting that classical features , such as perinuclear haloes associated with tumour cellularity , can predict genotype -LSB- 14 -RSB- . However , there are cases of histologically typical oligodendrogliomas without 1p 19q deletion and more astrocytic appearing tumours with deletion . For these cases clinical behaviour appears to be better predicted by genotype than phenotype , so that 1p 19q deletion provides additional information to conventional histopathological analysis -LSB- 27 -RSB- . Some studies suggest that oligodendrogliomas with 1p 19q deletion have lower activity of O6-methylguanine DNA methyltransferase -LRB- MGMT -RRB- due to promoter hypermethylation and decreased expression . MGMT is a DNA repair enzyme associated with resistance to alkylating agents , so this may contribute to chemosensitivity -LSB- 18 -RSB- , although other studies have not found a clear association -LSB- 42 -RSB- . At present , therefore , the biological basis for the intrinsically better prognosis of the 1p 19q deletion subgroup and the possible basis of a different interaction with adjuvant therapies remain poorly understood . In this study we hypothesise that the difference in behaviour of tumours with and without these deletions is due to differences in kinetic properties of the tumours related to proliferation and apoptosis . Apoptotic bodies are often a conspicuous feature in oligodendrogliomas and levels of apoptosis increase with grade -LSB- 43 -RSB- . Tumour growth depends on the net effects of cell proliferation and loss -LSB- 35 -RSB- so that a subgroup of tumours with a greater propensity to apoptosis versus proliferation may have slower growth . It is thus possible that increased susceptibility of tumour cells to undergo apoptosis might underlie both a better intrinsic biological behaviour and a greater sensitivity to adjuvant therapy . We hypothesise therefore that tumours with 1p ,19 q deletions show a greater tendency to apoptosis that is reflected in increased basal levels of apoptosis in the tumours . Differences in kinetic parameters related to DNA replication licensing are also candidates for relevance to growth capacity and chemosensitivity . The initiation of chromosomal replication is a crucial decision point in cell proliferation that lies at the point of convergence of all oncogenic signalling and transduction pathways that trigger proliferation . DNA replication initiation is precisely controlled through a core set of licensing factors -LRB- Orc1 -- 6 , Cdc6 , Cdt1 , Mcm2 -- 7 -RRB- that sequentially assemble into pre-replicative complexes -LRB- pre-RCs -RRB- at ∼ 30,000 replication origins scattered along the chromosomes , resulting in chromatin being ` licensed ' for replication in the subsequent S phase . Licensed replication origins are activated by the concerted action of S phase promoting kinases -LRB- Cdk2 -RRB- and the ASK-dependent Cdc7 kinase in a process commonly known as ` origin firing ' . Origin firing results in local melting of the DNA helix at replication origins and the recruitment of DNA polymerases -LRB- reviewed in -LSB- 40 -RSB- -RRB- . Notably the Mcm2 -- 7 replication licensing factors , which have been shown to act as a replicative DNA helicase , are present within the nucleus in cells in-cycle , but are down-regulated in out-of-cycle states -LSB- 10 , 38 -RSB- . We have previously shown that higher grade oligodendrogliomas show a greater proportion of cells that are licensed for DNA replication , as determined by the Mcm2 labelling index , which is of prognostic significance -LSB- 44 -RSB- . Certain tissues contain a population of licensed but non-proliferating cells -LSB- 10 , 38 -RSB- and higher labelling indices for Mcm2 than for the conventional proliferation marker Ki67 -LRB- which marks all four phases of the cell-cycle -RRB- suggest that this may be true for oligodendrogliomas -LSB- 44 -RSB- . A population of licensed but non-proliferating cells might conceivably resist cell-cycle dependent cytotoxicity and form a viable cell cache for tumour re-population . We have used the difference between the Mcm2 and Ki67 labelling indices -LRB- Mcm2 − Ki67 LI -RRB- as an assay of this population . It is essential for faithful propagation of the genome that origins are fired once and only once in each cell cycle to avoid re-replication events , which would lead to gene amplification . Geminin , a 23.5 kDa nuclear protein , is an endogenous regulator of chromosomal replication , absent during G1 phase but allowed to accumulate during S , G2 and M phase -LSB- 17 -RSB- . Geminin acts to prevent a second , inappropriate firing of replication origins by competitively binding to Cdt1 and thereby blocking recruitment of the Mcm2 -- 7 helicase to replication origins -LSB- 39 , 48 -RSB- . Thus pre-RC re-assembly and firing are prevented . At the metaphase to anaphase transition in mitosis , the anaphase promoting complex -LRB- APC/C -RRB- becomes activated and geminin is targeted for polyubiquitination and degradation via the 26S proteosome pathway , lowering levels to the point where the protein no longer interferes with assembly of pre-RCs during the following G1 phase -LSB- 17 -RSB- . Since geminin is present in S -- G2 -- M phase , the ratio of geminin to Ki67 may provide an index of the relative length of G1-phase . We have previously shown that this ratio decreases in higher-grade oligodendrogliomas , which may be a reflection of a shortened G1 in anaplastic tumours -LSB- 45 -RSB- . The length of G1-phase may be relevant to chemotherapeutic agents targeting the cell cycle machinery . We have therefore determined whether cytogenetic subgroups of oligodendrogliomas differ in their geminin/Ki67 ratio . Materials and methods A total of 55 cases of oligodendroglioma were identified and paraffin blocks retrieved from the archives of the Histopathology Department of the Royal Hallamshire Hospital , Sheffield , from a 12 year period from 1985 to 1997 . As previously described -LSB- 45 -RSB- , 25 cases were graded as WHO grade II -LRB- mean age 37.4 years -RRB- and 30 as grade III -LRB- mean age 44.6 years -RRB- . The series comprised 22 female and 33 male patients . All of the cases were resection specimens ; stereotactic and other small biopsies were excluded . Of these cases , 47 were first resections . Eight cases were recurrent resections that fell within the study period ; the initial resections on these cases were prior to the study period and not included . All of the cases were used for the labelling index analyses but only the 47 first resections were used for survival analysis . Clinical follow-up data and data on radiotherapy and chemotherapy were obtained from the Clinical Oncology Department at Western Park Hospital , Sheffield . Approval for the study was granted by the Local Research Ethics Committee . Immunohistochemistry Immunohistochemistry was performed on serial sections from a representative paraffin block from each case using a standard ABC method and the signal visualised using diaminobenzidine . Immunostaining was performed to Ki67 -LRB- Novocastra -RRB- , Mcm2 -LRB- BD Transduction Laboratories -RRB- and geminin -LRB- G95 , rabbit polyclonal -RRB- on this series of tumours as previously described -LSB- 45 -RSB- . Apoptotic bodies were counted on haematoxylin and eosin stained sections . Areas of highest cellularity were used for quantitation . The percentage of labelled cells , derived from a count of at least 1000 cells , was determined for each marker using an eyepiece graticule -LSB- 44 , 45 -RSB- . Fluorescence in situ hybridisation Fluorescence in situ hybridisation -LRB- FISH -RRB- was performed on 4 μ thick sections using the technique previously described -LSB- 7 -RSB- . The Vysis dual colour probe sets LSI 1p36/LSI 1q25 and LSI 19p13/LSI 19q13 were used according manufacturers instructions ; probes were hybridised overnight using a PTC Peltier Thermal Cycler . Analysis was performed independently by two scientists , both scoring 30 cells for each probe set . Statistical analyses Statistical analyses were performed using the statistical package for the social sciences -LRB- SPSS v10 .1 -RRB- . Comparison of age was made using an unpaired t test . Data on labelling indices was either not normally distributed and/or did not satisfy criteria for equality of variance between groups -LRB- Levene 's test -RRB- . Therefore comparisons of labelling indices between groups were performed using the non-parametric Mann -- Whitney U test . Fisher 's exact test was used for assessment of distribution between groups . For comparison of survival between groups , Kaplan Meier plots were constructed and the log rank test used . Multivariable linear regression analysis was used to examine the relationship of apoptotic index -LRB- AI -RRB- to proliferation and deletion status . Ki67 labelling index -LRB- LI -RRB- was used as measure of the former , rather than Mcm2 LI , as the latter also includes replication licensed but non-proliferating cells . To model a difference in the slope of the relationship between AI and Ki67 LI in the two subgroups , an interactive term between Ki67 LI and deletion status was included -LSB- Model : AI = β0 + -LRB- β1 × Ki67 LI -RRB- + -LRB- β2 × deletion status -RRB- + -LRB- β3 × Ki67 LI × deletion status -RRB- -RSB- . Results Characteristics of the deletion sub-groups FISH analysis for 1p and 19q deletion was successfully performed in 54 out of 55 cases -LRB- Fig. 1 -RRB- . Cases were assigned to the Del + group if they had deletion of both 1p and 19q , or 1p alone . Cases were assigned to the Del − group if they had no deletion or deletion of 19q only . There were 34 Del + cases and 20 Del − -LRB- Table 1 -RRB- . The Del + group had an older mean age than the Del − group -LRB- Table 2 -RRB- -LRB- P = 0.002 -RRB- . There was no difference in the distribution of histological grade between the two groups -LRB- P = 1 -RRB- . Fig. 1Representative FISH images using the VYSIS dual colour probe sets ; a relative loss of 1p -LRB- red -RRB- to 1q -LRB- green -RRB- with probable increase of chromosome number , b simple deletion of 1p -LRB- red -RRB- , c relative loss of 19q -LRB- red -RRB- to 19p -LRB- green -RRB- with probable increase of chromosome number , d simple deletion of 19q -LRB- red -RRB- Table 1Results of FISH analysisGroupDeletionNumberNo . in Del group % of casesDel +1 p 19q3434631p 0Del − None17203719q3Table 2Charateristics of cytogenetic sub-groupsGroupDel + Del − Mean age , SD46 .0 , 11.5 33.1 , 17.9 Grade II169Grade III1811 % of grade II47 .145.0 Survival analysis Survival for the 47 cases included in the survival analysis was taken from the date of the surgical resection . The eight -LRB- recurrent -RRB- cases excluded from this analysis were distributed evenly between Del + and Del − groups . The Del + group showed a trend to better survival -LRB- Fig. 2 -RRB- but this did not reach significance -LRB- P = 0.092 -RRB- , probably due to the number of censored cases . Further survival analysis to include multivariate parameters was therefore not attempted . Fig. 2Kaplan Meier survival curves for Del + -LRB- solid line -RRB- and Del − -LRB- dashed line -RRB- oligodendroglioma subgroups . Censored cases shown as cross marks Comparison of indices between cytogenetic subgroups The labelling index for apoptotic bodies -LRB- AI -RRB- , assessed from H&E stained sections was used as a measure of apoptosis . Higher levels of apoptosis were seen in the Del + group , carrying either deletions of 1p and 19q , or 1p alone -LRB- P = 0.037 -RRB- -LRB- Table 3 , Fig. 3 -RRB- . To further analyse the difference in apoptosis between the two cytogenetic subgroups , we considered whether higher levels of apoptosis in the Del + group were a reflection of higher cell turnover . However , there was no difference in replication-licensed or proliferating cells between the two subgroups as assessed by Mcm2 -LRB- P = 0.87 -RRB- or Ki67 -LRB- P = 0.77 -RRB- , respectively . Inspection of scatterplots of AI against Ki67 labelling index for the two subgroups suggested that with increasing tumour proliferation , apoptosis shows a greater increase in the Del + subgroup -LRB- Fig. 4 -RRB- . Because this suggested a steeper relationship of AI to proliferation in the Del + subgroup , multivariable regression analysis was carried out with a model , incorporating Ki67 LI , deletion status and a term for interaction between these two variables as predictors of apoptosis index . This gave an overall significance of P = 0.002 for the model . The interaction between Ki67 LI and deletion status was significant -LRB- P = 0.031 -RRB- and AI rose more steeply against Ki67 LI in the Del + group -LRB- AI α 0.58 xKi67LI -RRB- than in the Del − group -LRB- AI α 0.13 Ki67 LI -RRB- . Table 3Descriptive data for kinetic measuresSubgroupMeasureAIMcm2 − Ki67Gem/Ki67Del − Mean -LRB- SD -RRB- 0.72 -LRB- 0.38 -RRB- 8.31 -LRB- 22.21 -RRB- 0.39 -LRB- 0.25 -RRB- Median -LRB- IQR -RRB- 0.60 -LRB- 0.48 -RRB- 6.65 -LRB- 23.18 -RRB- 0.38 -LRB- 0.44 -RRB- Del + Mean -LRB- SD -RRB- 1.10 -LRB- 0.69 -RRB- 12.51 -LRB- 15.11 -RRB- 0.29 -LRB- 0.15 -RRB- Median -LRB- IQR -RRB- 0.95 -LRB- 1.00 -RRB- 7.15 -LRB- 21.88 -RRB- 0.26 -LRB- 0.22 -RRB- AI apoptotic index , Mcm2 − Ki67 Mcm2 minus Ki67 labelling index , Gem/Ki67 ratio of geminin to Ki67 indices , SD standard deviation , IQR interquartile rangeFig . 3Boxplot showing apoptotic indices in Del − and Del + subgroupsFig . 4Scatterplots of apoptosis index -LRB- AI -RRB- versus Ki67 labelling index for Del − -LRB- left -RRB- and Del + -LRB- right -RRB- cases The difference between Mcm2 and Ki67 labelling indices -LRB- Mcm2 − Ki67 LI -RRB- was used as a measure of the fraction of tumour cells that were licensed but not proliferating . In general tumours showed a higher labelling index for Mcm2 than Ki67 , indicating the presence of a population of licensed cells that are not actively replicating . The Mcm2 − Ki67 LI was higher in grade III than in grade II tumours -LRB- P = 0.001 , Fig. 5 -RRB- . However , we demonstrated no difference in the proportion of licensed , non-cycling cells by this method between the two cytogenetic subgroups of oligodendrogliomas -LRB- P = 0.46 -RRB- . We also demonstrated no difference in the geminin/Ki67 ratio -LRB- P = 0.22 -RRB- . Fig. 5Boxplot of Mcm2 minus Ki67 labelling index according to histological grade Discussion The basis for the difference in clinical behaviour between oligodendrogliomas with deletions of 1p and 19q -LRB- Del + -RRB- and those without -LRB- Del − -RRB- remains unclear , but its elucidation may yield important new avenues for improved therapeutic approaches . In this study we have investigated whether there are differences in parameters related to tumour cell proliferation and death in these two cytogenetic subgroups of tumours . The tumours were typed using FISH with probes to 1p and 19q . In our series , 63 % of cases showed deletions in 1p and 19q , or 1p alone , a similar proportion to that reported in series of oligodendrogliomas in the literature where figures for combined loss from approximately 50 to 70 % of cases have been described -LRB- sometimes higher in classical appearing WHO grade II tumours -RRB- -LSB- 13 , 19 , 24 , 25 , 33 -RSB- . Del + and Del − had a similar distribution of histological grades whilst the older average age in the Del + group is also unlikely to be of relevance to our findings as age is in general associated with a worse clinical behaviour , and opposite to the effects here . A trend towards better overall survival in Del + cases in our series did not reach significance , probably due to the number of total and censored cases . Analysis of the FISH probes in this cohort of cases revealed a pattern of multiple signals in some cases , indicating that increased copy number of chromosomes 1 and 19 were present , but that there was still effective loss of heterozygosity of short arm chromosome 1 and long arm chromosome 19 . -LRB- Fig. 1 -RRB- These cases were interpreted Del + . One other subgroup was identified in the cohort : three cases in which there was 19q deletion present without concomitant 1p deletion . There was no significant association of these subgroups in our series with an obvious pathological feature , but more cases should be studied to determine any clinico-pathological correlation . The multiple signal cases , which would indicate complex aneuploid karyotypes , usually found in tumours with more malignant status , may be of particular interest . One other group has identified the single 19q deletion status cases and have suggested that 19q deletion alone may also predict a favourable prognosis -LSB- 22 -RSB- . We have previously reported values for apoptosis , Mcm2 , Ki67 and geminin in this series of tumours -LSB- 45 -RSB- , but we now define indices and relate these to cytogenetic subtype . We have investigated three selected parameters that we hypothesise may affect survival and therapeutic response . Apoptosis is an important process in tumour biology as defective apoptosis induction may contribute to cancer development and the susceptibility of tumour cells to apoptosis may affect rate of growth . Apoptosis may be induced by adjuvant therapies and is likely to become an increasingly important molecular therapeutic target as specific pathways are defined -LRB- reviewed in -LSB- 26 -RSB- -RRB- . Apoptosis is variable between tumour types and a number of factors may contribute to apoptosis induction in a tumour . The apoptotic index often appears to be a function of the proliferation rate , with higher levels in more rapidly cycling tumours . In oligodendrogliomas , we and others have previously shown that apoptosis tends to increase with proliferation and with tumour grade -LSB- 29 , 43 -RSB- , though it has not been shown to be an independent prognostic factor . A similar relationship of apoptosis to histological grade and proliferation has also been demonstrated in the diffuse astrocytomas -LSB- 8 -RSB- . Although quantification of apoptosis , and so comparison between tumour types and studies , is beset with methodological difficulties , there is evidence that oligodendrogliomas show higher levels of apoptosis than other brain tumours of comparative WHO grade -LSB- 15 , 28 -RSB- . Alterations in regulatory factors related to molecular genetic changes are likely to be important in setting the susceptibility to apoptosis in a given tumour type , whilst other factors such as ischaemia and the influence of tumoural inflammatory cells may also contribute to apoptosis induction . Thus variation in apoptosis is a candidate to explain differences in clinical behaviour of tumour subtypes . In this series of oligodendrogliomas we have assessed apoptosis using counts of apoptotic bodies . This method has been used for other tumour types , including astrocytomas -LSB- 8 -RSB- . In a previous oligodendroglioma series we showed that the apoptotic index correlates with an assessment of apoptosis using the TUNEL method -LSB- 43 -RSB- , but it is a much simpler method . In addition , the TUNEL technique can be capricious and lacks specificity as it may detect DNA damage in cells from processes other than apoptosis , including necrosis -LSB- 12 -RSB- . Immunohistochemical detection of caspase 3 has also been used to detect apoptotic cells in histological sections -LSB- 5 -RSB- . We have previously carried out caspase 3 immunohistochemistry on this series -LSB- 45 -RSB- , but this showed weaker relationships to grade and proliferation than apoptotic index using counts of apoptotic bodies . Therefore , for this study , apoptotic index based on counts of apoptotic bodies was selected a priori for analysis . We have demonstrated that Del + oligodendrogliomas show a higher apoptotic index than Del - tumours , supporting the hypothesis that tumour cells from Del + cases may have a greater susceptibility to undergo apoptosis . We have not investigated the molecular pathways that might lead to this difference . However , it is not simply a reflection of increased cell turnover as Del + cases show no increase in either Mcm2 or Ki67 labelling indices compared to Del - cases -LRB- although a difference in cell-cycle length is not formally excluded -RRB- . The greater rate of increase in apoptotic index against Ki67 labelling index for Del + than Del - tumours suggests that oligodendrogliomas with 1p , 19q deletion have a greater tendency to the induction of apoptosis at higher rates of proliferation . Immunohistochemical demonstration and quantification of components of the DNA replication licensing machinery have recently been used for prognostic -LSB- 16 , 30 , 32 , 44 -RSB- and surveillance -LSB- 37 , 46 , 47 -RSB- studies in human tumours . Theoretically demonstration of MCM proteins may be considered superior to Ki67 as it identifies not only cycling cells , but also those non-cycling cells with proliferative potential -LSB- 10 , 38 -RSB- , and this superiority is born out in many of these studies . Combined assessment of these markers with other cell cycle related markers may be used to obtain information on the relative length of cell cycle phases in archival biopsy material -LSB- 9 , 20 , 31 -RSB- . In this study , which adds to these applications , we have used a variable generated from the difference between the Mcm2 and Ki67 labelling indices -LRB- Mcm2-Ki67 LI -RRB- for each case to assess the proportion of tumour cells that are licensed to replicate but not actually in cycle . This index has been previously used in renal cell cancer , where it increases with tumour grade -LSB- 6 -RSB- . We now also demonstrate that this index increases with tumour grade in oligodendrogliomas , suggesting that the pool of cells that are licensed but not proliferating is larger in more anaplastic tumours , evidence for greater dysregulation of the origin licensing pathway with anaplasia . Such a licensed , non-proliferating fraction might conceivably act as a treatment-resistant population that could re-grow after adjuvant therapy . There is increasing evidence for tumour stem cells in brain tumours -LRB- reviewed in -LSB- 11 -RSB- -RRB- and it is intriguing to speculate whether a stem cell population , responsible for perpetuating tumour growth , might reside in this fraction . Its assay is therefore of potential interest and we hypothesised that the size of this fraction might be greater in Del - tumours . However , we did not demonstrate any difference in Mcm2 − Ki67 LI between Del + and Del − tumours , suggesting that this is not important in determining their clinical differences . We have previously shown that the ratio of geminin to Ki67 labelling indices is increased in grade III versus grade II oligodendrogliomas -LSB- 45 -RSB- , implying a shortened G1-phase in higher grade tumours -LRB- assuming that other cell cycle phases are not substantially altered in duration -RRB- . In this study , however , we have not demonstrated evidence for a differing G1-phase duration between Del + and Del − tumours . Molecular differences between these two cytogenetic subtypes of oligodendroglioma are beginning to be defined , which will allow therapeutically relevant molecular classification -LSB- 24 -RSB- . Some of these genetic differences likely relate to the regulation of apoptosis , and may differ in the two subgroups . This study has shown that those oligodendrogliomas with 1p ± 19q deletions have higher apoptotic indices than those without . Differences in cell death susceptibility may affect prognosis through a treatment independent effect on intrinsic growth rate . But apoptosis may also be induced by adjuvant therapies , and amongst the gliomas this may be particularly important for oligodendroglial tumours -LSB- 36 -RSB- . This study does not allow distinction between intrinsic growth rate and treatment response contributions of apoptosis . It has also been limited to histological methods and has not provided functional data on susceptibility or defined its molecular basis . However , the findings support the hypothesis that differences in clinical outcomes between these cytogenetic subgroups is related to differing susceptibility to apoptosis induction and suggest that detailed comparative studies of apoptotic pathways in molecular subgroups of oligodendrogliomas are warranted .

O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O B O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O 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oligodendroglioma apoptosis replication cytogenetic