Medulloblastomas

overexpress

the

oncogene

Medulloblastoma

is

the

most

common

malignant

brain

tumor

of

childhood

Despite

numerous

advances

clinical

challenges

range

from

recurrent

and

progressive

disease

to

long-term

toxicities

in

survivors

The

lack

of

more

effective

less

toxic

therapies

results

from

our

limited

understanding

of

medulloblastoma

growth

Although

is

the

most

commonly

altered

gene

in

cancers

it

is

rarely

mutated

in

medulloblastoma

Accumulating

evidence

however

indicates

that

pathways

are

disrupted

in

medulloblastoma

phosphatase

-LRB-

or

-RRB-

encodes

a

negative

regulator

of

amplification

-LRB-

-RRB-

and

its

overexpression

have

been

reported

in

diverse

cancer

types

We

examined

primary

medulloblastoma

specimens

and

cell

lines

and

detected

copy

gain

and

amplification

prevalent

among

but

not

exclusively

in

the

tumors

with

gain

and

isochromosome

-LRB-

-RRB-

which

are

among

the

most

common

cytogenetic

lesions

in

medulloblastoma

RNA

levels

were

significantly

higher

in

the

tumors

with

gain

or

Immunoblots

confirmed

significant

protein

in

primary

tumors

generally

higher

in

those

with

gain

or

Under

basal

growth

conditions

and

in

response

to

the

chemotherapeutic

agent

etoposide

antagonized

apoptosis

in

medulloblastoma

cell

lines

These

results

indicate

that

medulloblastoma

express

significant

levels

of

that

modulate

genotoxic

responsiveness

by

negatively

regulating

Introduction

Medulloblastoma

is

the

most

common

malignant

brain

tumor

of

childhood

-LSB-

-RSB-

Treatment

with

surgery

radiation

and

chemotherapy

successfully

cures

many

patients

but

survivors

can

suffer

significant

long-term

toxicities

affecting

their

neurocognitive

and

growth

potential

-LSB-

-RSB-

Despite

clinical

advances

up

to

of

children

with

medulloblastoma

experience

tumor

progression

or

recurrence

for

which

no

curative

therapy

exists

The

lack

of

more

effective

less

toxic

therapies

stems

from

our

imperfect

understanding

of

the

molecular

processes

that

underlie

medulloblastoma

growth

Among

the

most

common

cytogenetic

lesions

affecting

medulloblastoma

are

the

gain

of

the

long

arm

of

chromosome

-LRB-

-RRB-

and

isochromosome

-LRB-

-RRB-

consisting

of

deletion

with

duplication

of

in

approximately

one-third

of

cases

-LSB-

-RSB-

Because

has

been

described

as

the

sole

cytogenetic

lesion

in

certain

medulloblastomas

it

may

represent

a

primary

event

rather

than

an

alteration

associated

with

clonal

evolution

-LSB-

-RSB-

Investigators

have

long

sought

oncogenes

on

and

tumor

suppressor

genes

on

but

with

limited

success

-LSB-

-RSB-

Although

the

tumor

suppressor

gene

-LRB-

-RRB-

is

mutated

in

approximately

half

of

human

malignancies

it

is

rarely

mutated

in

medulloblastoma

-LSB-

-RSB-

However

several

lines

of

evidence

suggest

that

the

pathway

is

perturbed

in

medulloblastoma

Frank

et

al.

have

described

activation

of

the

pathway

in

the

large

cell/anaplastic

variant

of

medulloblastoma

-LSB-

-RSB-

Our

collaborators

have

noted

significant

nuclear

immunopositivity

consistent

with

its

activation

in

approximately

of

primary

human

medulloblastoma

examined

-LRB-

Adekunle

Adesina

personal

communication

-RRB-

Deletion

of

the

murine

homolog

in

the

Patched

haploinsufficient

-LRB-

Ptch

-RRB-

mouse

model

increases

the

incidence

of

spontaneous

medulloblastoma

from

approximately

to

-LSB-

-RSB-

Individuals

with

Li

Fraumeni

syndrome

who

carry

germline

mutations

are

at

increased

risk

for

developing

medulloblastoma

but

fewer

than

of

sporadic

medulloblastoma

display

mutations

-LSB-

-RSB-

The

activity

of

may

be

abrogated

by

alternate

mechanisms

as

observed

in

other

cancers

with

-LSB-

-RSB-

One

mechanism

by

which

function

is

limited

is

through

amplification

or

overexpression

of

encodes

an

ubiquitin

ligase

that

normally

functions

in

a

negative

feedback

loop

to

down-regulate

expression

and

is

amplified

in

approximately

of

human

malignancies

that

lack

mutation

of

-LSB-

-RSB-

However

Adesina

et

al.

and

Batra

et

al.

have

documented

the

absence

of

amplification

in

medullobastoma

-LSB-

-RSB-

Although

overexpression

has

been

detected

in

adult

patients

-LSB-

-RSB-

there

is

no

functional

data

to

implicate

in

inactivation

in

medulloblastoma

suggesting

alternative

mechanisms

The

proto-oncogene

-LRB-

phosphatase

or

protein

phosphatase

magnesium-dependent

delta

-RRB-

maps

to

Amplification

of

has

been

described

in

several

cell

lines

and

malignancies

including

neuroblastoma

breast

and

ovarian

carcinomas

most

of

which

are

wild-type

for

-LSB-

-RSB-

displays

oncogenic

properties

by

inactivating

and

ATM

pathways

-LSB-

-RSB-

Previous

reports

of

genomic

analysis

of

medulloblastoma

have

implicated

the

gain

of

the

locus

in

its

overexpression

in

a

subset

of

tumors

-LSB-

-RSB-

We

present

data

that

confirm

prior

observations

and

also

define

the

effects

of

on

medulloblastoma

cell

growth

We

have

quantitated

overexpression

in

primary

medulloblastoma

specimens

and

cell

lines

previously

analyzed

by

comparative

genomic

hybridization

-LRB-

CGH

-RRB-

-LSB-

-RSB-

Using

fluorescence

in

situ

hybridization

-LRB-

FISH

-RRB-

we

determined

that

copy

gain

and

amplification

is

prevalent

among

but

not

exclusively

found

in

the

tumors

with

gain

and

We

surveyed

primary

medulloblastoma

specimens

for

expression

and

determined

that

RNA

levels

were

significantly

higher

in

tumors

with

gain

or

Similarly

our

Western

immunoblot

analysis

confirmed

significant

levels

of

protein

expression

in

primary

tumors

generally

higher

in

those

with

gain

or

Genotoxic

stress

induces

phosphorylation

and

expression

in

and

Daoy

medulloblastoma

cell

lines

Transient

transfections

indicate

that

antagonizes

cell

cycle

arrest

and

apoptosis

in

response

to

the

chemotherapeutic

agent

etoposide

These

results

support

the

hypothesis

that

medulloblastoma

express

significant

levels

of

that

modulate

genotoxic

responsiveness

by

negatively

regulating

Materials

and

methods

Medulloblastoma

tissue

and

cell

lines

Medulloblastoma

samples

-LRB-

n

-RRB-

were

obtained

from

children

diagnosed

at

Texas

Children

's

Hospital

Houston

TX

between

and

following

informed

consents

Tissues

were

snap-frozen

and

stored

in

liquid

nitrogen

until

processed

All

specimens

were

obtained

at

the

time

of

diagnosis

prior

to

radiation

or

chemotherapy

and

subjected

to

histopathologic

review

according

to

WHO

criteria

-LSB-

-RSB-

Seven

tumors

displayed

classic

histology

were

uniformly

desmoplastic

nodular

or

well-differentiated

seven

were

predominantly

anaplastic

or

large

cell

an

additional

seven

displayed

some

degree

of

anaplasia

or

large

cell

histology

and

one

was

regarded

as

of

the

above

Ten

patients

were

female

and

were

male

The

mean

age

at

diagnosis

was

months

-LRB-

median

months

-RRB-

with

four

patients

less

than

months

old

-LRB-

range

months

-RRB-

The

median

and

mean

time

of

follow-up

was

approximately

months

from

diagnosis

The

majority

of

patients

were

-LRB-

non-metastatic

-RRB-

while

one

was

and

eight

were

stage

at

diagnosis

All

studies

were

performed

according

to

the

institutional

guidelines

of

Texas

Children

's

Hospital

and

with

the

approval

of

the

Institutional

Review

Board

of

Baylor

College

of

Medicine

The

breast

carcinoma

cell

line

with

known

high-level

amplification

the

un-amplified

breast

epithelial

cell

line

and

the

medulloblastoma

cell

lines

Daoy

and

-LRB-

American

Type

Culture

Collection

Manassas

VA

-RRB-

were

maintained

as

previously

described

in

Dulbecco

's

modified

Eagle

's

medium

-LRB-

Invitrogen

Carlsbad

CA

-RRB-

with

high

glucose

-LRB-

g/l

-RRB-

mM

l-glutamine

and

-LRB-

vol/vol

-RRB-

heat-inactivated

fetal

calf

serum

-LRB-

Invitrogen

Carlsbad

CA

-RRB-

at

°C

in

Etoposide

-LRB-

-RRB-

and

cisplatin

-LRB-

CDDP

-RRB-

were

applied

to

cell

cultures

as

media-diluted

stock

solutions

in

PBS

and

DMSO

respectively

-LRB-

Sigma

St.

Louis

MO

-RRB-

Ultraviolet

-LRB-

UV

-RRB-

irradiation

was

performed

by

exposing

cell

cultures

using

a

GS

Gene

Linker

UV

Chamber

-LRB-

Bio-Rad

Laboratories

Hercules

CA

-RRB-

Fluorescence

in

situ

hybridization

-LRB-

FISH

-RRB-

analysis

Primary

medulloblastoma

cells

were

harvested

from

first

passage

cultures

Chromosomes

were

prepared

according

to

standard

methods

including

confirmatory

karyotyping

with

G-banding

-LSB-

-RSB-

Eleven

first

or

second

passage

cultures

of

primary

tumors

were

harvested

when

cultures

were

still

sub-confluent

The

BAC

containing

the

locus

at

chromosome

-LRB-

-RRB-

and

a

plasmid

containing

chromosome

centromeric

sequences

-LRB-

generously

provided

by

Dr.

Mariano

Rocchi

-RRB-

were

fluorescently

labeled

with

Spectrum

Green

and

Spectrum

Orange

respectively

-LRB-

Vysis

Downers

Grove

IL

-RRB-

by

nick

translation

and

hybridized

to

metaphase

or

interphase

spreads

of

cell

lines

-LRB-

Daoy

-RRB-

and

primary

tumor

specimens

-LSB-

-RSB-

We

confirmed

the

map

position

of

on

normal

human

metaphase

spreads

by

FISH

as

described

previously

-LSB-

-RSB-

The

slides

were

counterstained

with

DAPI

and

the

images

were

captured

using

the

Quips

Pathvysion

System

-LRB-

Applied

Imaging

Santa

Clara

CA

-RRB-

To

determine

the

copy

number

status

we

analyzed

individual

interphase

nuclei

for

each

case

The

presence

of

copy

gain

or

amplification

was

indicated

if

more

than

of

tumor

nuclei

displayed

an

increased

copy

number

relative

to

the

chromosome

centromeric

probe

signals

and/or

tumor

ploidy

In

cases

in

which

there

was

a

discrepancy

between

the

number

of

centromeric

signals

and

ploidy

the

ploidy

number

was

used

to

estimate

the

relative

copy

number

Quantitative

real-time

RT-PCR

-LRB-

qRT-RTPCR

-RRB-

analysis

Total

cellular

RNA

was

prepared

from

frozen

tumor

tissue

with

either

Trizol

-LRB-

Invitrogen

-RRB-

or

an

RNeasy

kit

-LRB-

Qiagen

Valencia

CA

-RRB-

depending

upon

relative

abundance

according

to

the

manufacturers

recommendations

Integrity

and

concentration

of

RNA

were

verified

on

the

Baylor

College

of

Medicine

Houston

TX

Microarray

Core

Facility

Bioanalyzer

-LRB-

Agilent

Technologies

Santa

Clara

CA

-RRB-

Total

cellular

RNA

was

reverse

transcribed

with

SuperScript

First-Strand

Synthesis

-LRB-

Invitrogen

-RRB-

primed

with

oligo

-LRB-

dT

-RRB-

PCR

reactions

were

performed

in

a

Bio-Rad

iCycler

using

iQ

Syber

Green

Supermix

-LRB-

Bio-Rad

Laboratories

-RRB-

and

primers

listed

below

Each

PCR

reaction

was

performed

for

cycles

and

performed

in

triplicate

Primer

sequences

included

sense

and

antisense

sense

and

antisense

GAPDH

sense

and

antisense

Expression

of

target

RNA

was

internally

normalized

to

expression

of

GAPDH

gene

and

expressed

relative

to

target

gene

expression

in

a

human

fetal

brain

RNA

pool

-LRB-

Stratagene

La

Jolla

CA

-RRB-

Human

adult

cerebellar

-LRB-

Stratagene

-RRB-

and

human

fetal

cerebellar

RNA

samples

-LRB-

BioChain

Hayward

CA

-RRB-

were

also

assayed

to

provide

tissue

controls

Amplification

products

were

verified

by

analyzing

melting

curves

agarose

gel

electrophoresis

and

with

direct

sequencing

of

PCR

products

Western

immunoblot

analysis

Protein

was

extracted

from

frozen

tissue

by

solubilization

in

boiling

lysis

buffer

-LRB-

SDS

mM

Tris

Cl

pH

mM

β-mercaptoethanol

-RRB-

Lysates

were

stored

at

°C

and

separated

by

SDS-PAGE

transferred

to

nitrocellulose

membranes

-LRB-

Pall

Life

Sciences

Ann

Arbor

MI

-RRB-

and

immunoblotted

using

standard

methods

Primary

antibodies

included

monoclonal

antibody

-LRB-

Trevigen

Gaithersburg

MD

-RRB-

-LRB-

-RRB-

specific

monoclonal

antibody

-LRB-

Cell

Signaling

Technology

Danvers

MA

-RRB-

monoclonal

antibody

-LRB-

Santa

Cruz

Biotechnology

Santa

Cruz

CA

-RRB-

and

β-Actin

monoclonal

antibody

-LRB-

Sigma

-RRB-

Staining

with

secondary

antibody

-LRB-

Alexa

Fluor

goat

anti-mouse

IgG

Invitrogen

-RRB-

was

visualized

using

an

Odyssey

infrared

imaging

system

-LRB-

LI-COR

Biosciences

Lincoln

Nebraska

-RRB-

Intensity

of

immunostaining

was

quantitated

using

ImageQuant

-LRB-

Molecular

Dynamics

Piscataway

NJ

-RRB-

Expression

of

target

proteins

in

each

sample

was

internally

normalized

to

β-actin

-LRB-

determined

on

re-probed

blots

-RRB-

relative

to

expression

in

the

cell

line

Transfections

and

luciferase

reporter

assays

Cell

lines

were

transiently

transfected

with

expression

and

reporter

plasmids

using

Lipofectin

according

to

manufacturer

's

recommendations

-LRB-

Invitrogen

-RRB-

Expression

plasmids

included

the

human

cytomegalovirus

-LRB-

CMV

-RRB-

immediate-early

-LRB-

IE

-RRB-

promoter-driven

plasmid

encoding

a

green

fluorescent

protein

-LRB-

GFP

-RRB-

fusion

protein

-LRB-

Clontech

Mountainview

CA

-RRB-

Reporter

plasmids

included

the

construct

to

detect

transcription

-LSB-

-RSB-

and

the

actin

promoter-driven

Renilla

luciferase

plasmid

pB-Actin-RL

to

provide

transfection

controls

-LSB-

-RSB-

In

each

transfection

the

expression

and

reporter

plasmids

were

supplemented

by

empty

vector

plasmid

-LRB-

Invitrogen

-RRB-

to

achieve

an

equivalent

total

DNA

concentration

For

flow

cytometry

the

EGFP-expressing

pMaxGFP

plasmid

-LRB-

Amaxa

Gaithersburg

MD

-RRB-

provided

transfection

controls

For

functional

assessment

of

in

vitro

activation

cells

transfected

with

luciferase-encoding

reporter

plasmids

were

harvested

and

processed

for

dual

color

luciferase

assay

according

to

manufacturer

's

recommendations

-LRB-

Promega

Madison

WI

-RRB-

and

quantitated

using

a

BenchMark

Plus

plate

spectrophotometer

-LRB-

Bio-Rad

-RRB-

Flow

cytometric

and

terminal

deoxynucleotidyl

transferase-mediated

biotinylated-dUTP

nick

end-labeling

-LRB-

TUNEL

-RRB-

analysis

We

monitored

DNA-indices

for

cell

cycle

analysis

by

multi-parametric

flow

cytometry

using

standard

methods

Analyses

were

performed

using

a

Becton

Dickinson

FACScan

flow

cytometer

-LRB-

BD

Biosciences

San

Jose

CA

-RRB-

for

the

detection

of

cells

stained

with

propidium

iodide

-LRB-

PI

-RRB-

and

a

nm

laser

with

filter

combination

for

fluorescein

isothiocyanate

-LRB-

FITC

-RRB-

and

GFP

Single

cell

suspensions

were

isolated

from

culture

fixed

in

methanol

and

stained

with

PI

-LRB-

μg/ml

in

PBS

-RRB-

Each

histogram

represents

cells

for

measuring

DNA-index

and

cell

cycle

Histogram

analysis

was

performed

with

the

CellQuest

program

-LRB-

BD

Biosciences

-RRB-

We

calculated

the

peak

at

a

position

in

the

hypodiploid

area

below

a

DNA-index

of

one

-LRB-

-RRB-

Because

the

nucleus

becomes

fragmented

during

apoptosis

and

numerous

individual

chromatin

fragments

may

be

present

in

a

single

cell

the

percentage

of

objects

with

a

fractional

DNA-content

is

represented

by

the

peak

Apoptotic

nuclei

were

identified

as

a

sub-diploid

DNA

peak

and

were

distinguished

from

cell

debris

on

the

basis

of

forward

light

scatter

and

PI

fluorescence

The

significance

of

differences

in

apoptotic

populations

was

determined

using

paired

Student

t-test

To

detect

apoptotic

nuclei

in

peaks

a

subset

of

unstained

cells

was

analyzed

for

DNA-fragmentation

using

the

Frag-EL

DNA

fragmentation

detection

kit

according

to

manufacturer

's

recommendations

-LRB-

Oncogene

Research

Products

Cambridge

MA

-RRB-

Cells

were

also

counter-stained

with

PI

for

DNA

quantification

FITC

signal

was

detected

on

one

channel

in

the

logarithmic

mode

while

UV

fluorescence

-LRB-

PI

-RRB-

was

recorded

in

the

linear

mode

on

a

separate

channel

For

each

measurement

at

least

cells

were

analyzed

We

used

CellQuest

software

for

multi-parametric

calculations

and

analyses

Cutoff

negative

and

positive

cells

resulted

from

FITC-fluorescence

isotype

control

measurements

for

each

sample

Results

DNA

copy

number

is

increased

in

medulloblastomas

with

gain

We

employed

FISH

to

analyze

copy

numbers

of

the

gene

in

primary

medulloblastoma

specimens

and

cell

lines

previously

analyzed

by

CGH

-LRB-

Fig.

-RRB-

Compared

to

the

centromeric

chromosome

probe

which

consistently

displayed

signals

in

each

metaphase

or

interphase

nucleus

the

BAC

probe

-LRB-

-RRB-

containing

the

locus

revealed

a

wider

range

of

copy

numbers

By

FISH

we

identified

high-level

amplification

of

the

gene

-LRB-

copies/cell

-RRB-

in

all

of

the

cells

analyzed

-LRB-

Fig.

-RRB-

This

finding

is

consistent

with

previously

identified

amplification

of

the

chromosomal

region

in

cells

The

-LRB-

-RRB-

medulloblastoma

cell

line

with

displayed

low-level

amplification

of

the

locus

with

four

to

nine

copies

in

each

interphase

spread

examined

-LRB-

Fig.

-RRB-

In

contrast

the

Daoy

medulloblastoma

cell

line

with

trisomy

displayed

an

average

of

three

copies

of

-LRB-

Fig.

-RRB-

Fig.

copy

number

is

increased

in

medulloblastomas

with

gain

of

chromosome

High

power

micrographs

of

interphase

nuclei

of

cell

lines

and

primary

human

medulloblastoma

cells

hybridized

to

fluorescently-labeled

BAC

-LRB-

green

-RRB-

and

centromeric

chromosome

probes

-LRB-

red

-RRB-

-LRB-

A

-RRB-

nucleus

showing

numerous

amplicons

-LRB-

B

-RRB-

-LRB-

-RRB-

nucleus

exhibiting

low-level

amplification

-LRB-

C

-RRB-

Daoy

nucleus

showing

gain

of

-LRB-

D

L

-RRB-

interphase

nuclei

of

medulloblastoma

cells

from

nine

patients

illustrating

examples

of

-LRB-

D

H

-RRB-

diploid

and

near-diploid

nuclei

and

-LRB-

I

L

-RRB-

those

with

low-level

amplification

of

Among

the

tumors

analyzed

by

CGH

primary

medulloblastomas

were

examined

for

amplification

by

FISH

-LSB-

-RSB-

Six

tumors

displayed

two

showed

trisomy

one

had

loss

of

with

gain

of

and

two

had

no

change

in

chromosome

-LRB-

Table

-RRB-

We

detected

low-level

amplification

of

the

locus

in

of

primary

medulloblastoma

specimens

which

also

had

gain

of

including

four

with

by

G-banding

Table

amplification

is

associated

with

gain

of

chromosome

or

in

primary

medulloblastoma

specimensSpecimenCopy

number

-LRB-

FISH

-RRB-

Amplification

CNA

-LRB-

CGH

-RRB-

CEP

analysis

for

and

chromosome

centromere

reveals

low-level

amplification

of

in

of

primary

medulloblastoma

samples

copy

gain

is

significantly

associated

with

gain

of

-LRB-

P

-RRB-

Amplification

is

defined

as

copies

as

determined

by

FISH

Reportedly

amplified

but

not

determined

in

the

present

studyAbbreviations

MB

medulloblastoma

gain

of

chromosome

loss

of

chromosome

N/C

no

change

in

chromosome

CNA

copy

number

aberration

CEP

centromeric

chromosome

probe

Four

tumors

were

diploid

or

near-diploid

overall

-LRB-

Fig.

G

-RRB-

and

seven

displayed

copy

number

gain

of

the

locus

-LRB-

Fig.

L

-RRB-

Our

previously

reported

CGH

analysis

of

medulloblastoma

revealed

that

had

gain

of

including

with

and

with

amplifications

involving

consistent

with

previous

reports

-LSB-

-RSB-

Comparing

CGH

results

with

our

FISH

analysis

reveals

that

DNA

copy

number

correlates

with

gain

of

-LRB-

including

-RRB-

by

CGH

-LRB-

P

Student

's

t-test

-RRB-

All

the

tumors

with

copy

number

gain

of

by

FISH

also

had

gain

of

the

locus

on

chromosome

by

CGH

amplification

in

medulloblastoma

was

low-level

compared

to

the

high-level

amplification

observed

in

cells

These

cytogenetic

results

indicate

that

low-level

amplification

of

the

locus

are

prevalent

in

a

significant

proportion

of

medulloblastoma

Medulloblastomas

with

gain

of

including

overexpress

RNA

Using

qRT-RTPCR

we

determined

the

expression

of

RNA

in

cell

lines

and

a

larger

series

of

primary

medulloblastoma

samples

Human

fetal

cerebellum

specimens

displayed

greater

expression

-LRB-

SEM

-RRB-

compared

to

fetal

whole

brain

Human

adult

cerebellum

expresses

more

RNA

-LRB-

-RRB-

than

fetal

total

brain

cells

had

higher

levels

of

RNA

-LRB-

-RRB-

relative

to

human

fetal

brain

-LRB-

Fig.

-RRB-

Daoy

cells

expressed

RNA

-LRB-

SEM

-RRB-

compared

to

fetal

brain

-LRB-

Fig.

-RRB-

In

contrast

cells

displayed

-LRB-

-RRB-

higher

expression

of

RNA

than

human

fetal

brain

Fig.

with

gain

of

express

significantly

higher

RNA

levels

-LRB-

A

-RRB-

RNA

expression

by

qRT-RTPCR

analysis

is

significantly

higher

in

the

cell

line

and

in

primary

medulloblastoma

specimens

with

gain

of

or

-LRB-

MB

n

-RRB-

by

conventional

karyotyping

and

CGH

compared

to

Daoy

and

primary

tumors

without

or

gain

of

-LRB-

gain

MB

n

-RRB-

For

reference

expression

in

the

breast

cancer

cell

line

is

also

shown

Columns

mean

expression

of

at

least

three

experiments

error

bars

SEM

Y-axis

RNA

expression

normalized

to

GAPDH

expression

and

relative

to

human

fetal

brain

-LRB-

B

-RRB-

Relative

RNA

expression

in

individual

medulloblastoma

specimens

-LRB-

n

-RRB-

assayed

by

qRT-RTPCR

and

listed

with

their

associated

chromosome

status

-LRB-

C

-RRB-

Relative

RNA

expression

in

medulloblastomas

with

amplification

-LRB-

open

diamond

-RRB-

is

significantly

higher

than

non-amplified

tumors

-LRB-

open

circle

-RRB-

Abbreviations

ALL

MB

mean

-LRB-

SEM

-RRB-

of

all

primary

human

tumors

isochromosome

gain

of

chromosome

loss

of

chromosome

NC

no

change

in

chromosome

We

surveyed

primary

medulloblastoma

specimens

for

expression

and

determined

that

RNA

levels

were

significantly

higher

in

tumors

with

gain

or

-LRB-

Fig.

Table

-RRB-

Of

the

primary

medulloblastomas

examined

by

FISH

tumors

-LRB-

n

-RRB-

displayed

significantly

higher

RNA

levels

than

non-amplified

tumors

-LRB-

n

-RRB-

-LRB-

-LRB-

-RRB-

vs.

-LRB-

-RRB-

more

RNA

than

fetal

control

P

-RRB-

-LRB-

Fig.

Table

-RRB-

Among

the

larger

set

of

analyzed

specimens

primary

medulloblastoma

samples

displayed

higher

levels

of

RNA

-LRB-

-RRB-

than

human

fetal

brain

-LRB-

Fig.

B

Table

-RRB-

Analyzed

separately

tumors

with

gain

of

including

-LRB-

n

-RRB-

expressed

more

RNA

-LRB-

-RRB-

compared

to

those

without

gain

of

or

-LRB-

n

-RRB-

with

only

more

RNA

-LRB-

-RRB-

relative

to

human

fetal

brain

-LRB-

Fig.

B

Table

-RRB-

In

other

words

those

tumors

with

gain

of

including

expressed

more

RNA

-LRB-

-RRB-

than

those

without

such

cytogenetic

lesions

The

association

between

RNA

expression

and

the

presence

or

absence

of

gain

-LRB-

including

-RRB-

was

statistically

significant

-LRB-

P

-RRB-

-LRB-

Table

-RRB-

Table

overexpression

is

associated

with

gain

of

chromosome

or

in

primary

medulloblastoma

specimensTumors

grouped

by

RNAP

value

q

q

or

gain

Normal

copy

Tumors

analyzed

by

FISH

-LRB-

subtotal

-RRB-

q

q

or

gain

All

tumors

analyzed

by

CGH

-LRB-

total

-RRB-

amplification

by

FISH

was

associated

with

increased

RNA

expression

by

qRT-RTPCR

-LRB-

P

-RRB-

Relative

RNA

level

was

significantly

associated

with

gain

including

-LRB-

P

-RRB-

P

value

Student

t-test

of

tumors

with

specified

CNA

versus

tumors

without

that

CNA

P

value

Student

t-test

of

versus

non-amplified

tumorsAbbreviations

MB

medulloblastoma

gain

of

chromosome

loss

of

chromosome

CNA

copy

number

aberration

Medulloblastomas

with

gain

of

overexpress

protein

A

subset

of

representative

tumors

with

sufficient

tissue

was

analyzed

for

protein

expression

Western

immunoblot

analysis

confirmed

significant

protein

expression

in

lysates

from

medulloblastoma

cell

lines

and

tumor

samples

internally

normalized

to

β-actin

expression

and

relative

to

expression

in

the

cell

line

-LRB-

Fig.

B

-RRB-

The

medulloblastoma

cell

line

Daoy

with

trisomy

expressed

more

protein

-LRB-

SEM

-RRB-

than

cells

-LRB-

Fig.

B

-RRB-

The

cell

line

with

displayed

higher

levels

-LRB-

-RRB-

than

-LRB-

Fig.

B

-RRB-

Since

UV

irradiation

activates

by

phosphorylation

at

serine

and

induces

expression

-LSB-

-RSB-

we

used

lysates

prepared

from

irradiated

cells

to

provide

control

specimens

with

induced

and

proteins

In

UV-irradiated

cells

levels

stabilized

with

increased

phosphorylation

at

-LRB-

Fig.

-RRB-

The

breast

cancer

line

expressed

more

-LRB-

-RRB-

than

cells

-LRB-

Fig.

B

-RRB-

Fig.

express

significant

protein

-LRB-

A

-RRB-

Western

blots

of

cell

lines

and

four

primary

medulloblastoma

specimens

reveal

significant

and

protein

expression

UV

irradiation

-LRB-

-RRB-

of

increases

and

expression

and

induces

phosphorylation

detected

as

-LRB-

-RRB-

Breast

carcinoma

cell

lines

and

non-amplified

are

shown

for

reference

Associated

chromosome

lesions

are

also

labeled

-LRB-

B

-RRB-

Western

analysis

of

protein

expression

among

primary

medulloblastoma

specimens

-LRB-

n

-RRB-

normalized

to

internal

reference

β-actin

and

relative

to

expression

in

the

cell

line

Abbreviations

all

MB

mean

-LRB-

SEM

-RRB-

of

all

primary

human

tumors

-LRB-

n

-RRB-

isochromosome

gain

of

chromosome

loss

of

chromosome

CNA

copy

number

aberration

Of

the

ten

primary

medulloblastoma

specimens

analyzed

five

displayed

gain

of

or

one

had

trisomy

another

had

loss

of

and

three

showed

no

change

in

chromosome

by

CGH

Overall

mean

protein

expression

was

-LRB-

-RRB-

greater

than

expression

in

cells

internally

normalized

to

β-actin

-LRB-

Fig.

-RRB-

As

a

group

primary

tumors

exhibited

even

more

than

the

cell

line

which

is

widely

employed

as

an

example

of

Analyzed

separately

primary

medulloblastomas

with

or

-LRB-

n

-RRB-

displayed

-LRB-

-RRB-

more

protein

than

compared

to

gain

tumors

-LRB-

n

-RRB-

with

only

-LRB-

-RRB-

levels

-LRB-

Fig.

-RRB-

We

also

analyzed

primary

medulloblastoma

lysates

for

expression

of

total

protein

and

phosphorylation

at

serine

-LRB-

-RRB-

-LRB-

Fig.

-RRB-

Total

and

-LRB-

-RRB-

were

readily

detectable

on

Western

blots

and

suggest

functional

significance

Overall

Western

analysis

indicated

significant

levels

of

protein

expression

in

primary

tumors

generally

higher

in

those

with

gain

or

which

might

be

responsible

for

antagonizing

activation

Genotoxic

stress

induces

phosphorylation

and

expression

in

medulloblastoma

cell

lines

To

determine

the

functional

significance

of

overexpression

we

transiently

transfected

established

medulloblastoma

cell

lines

and

assayed

changes

in

cell

cycle

and

apoptosis

in

response

to

altered

levels

of

The

and

Daoy

cell

lines

differ

in

their

basal

expression

and

their

status

providing

cell

systems

for

gain-of-function

transfection

studies

Genotoxic

stress

in

the

form

of

exposure

to

UV

irradiation

or

the

chemotherapeutic

agent

stabilized

levels

and

induced

phosphorylation

of

in

and

Daoy

cells

respectively

-LRB-

Fig.

-RRB-

We

examined

the

effects

of

increased

constitutive

by

transient

transfection

with

a

flag-tagged

expression

plasmid

Flag-tagged

could

be

readily

distinguished

from

endogenous

on

Western

blots

and

transfection

was

accompanied

by

decreased

-LRB-

-RRB-

in

treated

and

Daoy

cells

that

are

wild-type

and

mutant

for

respectively

-LRB-

Fig.

-RRB-

Fig.

antagonizes

activation

in

medulloblastoma

cell

lines

-LRB-

A

-RRB-

Western

blots

of

and

Daoy

medulloblastoma

cell

lines

Treatment

with

UV

irradiation

-LRB-

UV

-RRB-

or

etoposide

-LRB-

μM

-RRB-

stabilizes

wild-type

protein

and

induces

phosphorylation

of

at

Transfection

with

the

expression

plasmid

detected

as

higher

molecular

weight

band

using

or

anti-flag

antibodies

Overexpressed

reduces

phosphorylation

at

serine

-LRB-

-RRB-

-LRB-

B

-RRB-

Increased

and

RNA

in

and

Daoy

cell

lines

transfected

with

and

-LRB-

-RRB-

expression

plasmids

compared

to

the

MaxGFP

control

plasmid

as

measured

by

qRT-RTPCR

-LRB-

C

-RRB-

Luciferase

assays

reveal

that

co-transfection

of

-LRB-

-RRB-

partially

abrogates

transcription

from

the

reporter

-LRB-

relative

to

constitutively

expressed

control

Renilla-luciferase

reporter

-RRB-

in

transfected

and

Daoy

cells

-LRB-

error

bars

SEM

-RRB-

-LRB-

D

-RRB-

TUNEL

assay

and

flow

cytometric

methods

confirm

apoptosis

in

Daoy

and

cell

lines

Determination

of

RNA

levels

with

qRT-RTPCR

confirmed

significant

expression

of

and

by

transfection

with

and

expression

plasmids

-LRB-

Fig.

-RRB-

To

confirm

the

transcriptional

effects

of

and

plasmids

we

co-transfected

cell

lines

with

the

reporter

plasmid

and

an

actin

promoter-driven

Renilla

luciferase

plasmid

to

control

for

transfection

efficiency

Transfected

partially

abrogated

luminescence

indicating

activity

in

both

cell

lines

in

vitro

Presumably

because

of

endogenous

wild-type

the

relative

response

in

cells

was

less

dramatic

than

in

Daoy

cells

-LRB-

Fig.

-RRB-

and

constitutively

expressed

induce

apoptosis

Expression

from

epitope-tagged

plasmids

enabled

us

to

employ

flow

cytometric

methods

to

analyze

the

cell

cycle

distribution

of

fluorescently

immunolabeled

and

transfectants

To

stimulate

genotoxic

responses

in

vitro

we

employed

minimally

toxic

dose

concentrations

of

etoposide

-LRB-

-RRB-

a

chemotherapeutic

topoisomerase

II-inhibitor

used

to

treat

medulloblastoma

clinically

changes

in

apoptosis

were

also

determined

by

flow

cytometric

quantitation

of

cells

in

fractions

which

were

confirmed

by

TUNEL

analysis

-LRB-

Fig.

-RRB-

Similar

results

were

obtained

with

micromolar

concentrations

of

another

chemotherapeutic

agent

cisplatin

-LRB-

data

not

shown

-RRB-

When

exposed

to

low

concentrations

of

the

medulloblastoma

cell

line

underwent

increased

apoptosis

indicated

by

an

increased

fraction

of

cells

in

by

flow

cytometric

analysis

from

to

-LRB-

P

-RRB-

-LRB-

Fig.

-RRB-

These

results

indicate

that

induces

apoptosis

as

described

for

other

cell

types

with

wild-type

Fig.

antagonizes

apoptosis

and

arrest

in

medulloblastoma

cell

lines

Flow

cytometric

analysis

of

cell

cycle

distributions

of

-LRB-

A

-RRB-

untreated

controls

illustrating

the

cell

populations

with

DNA

content

in

phase

DNA

content

in

phase

intermediate

DNA

content

in

S

phase

and

the

peak

representing

apoptotic

nuclei

-LRB-

B

-RRB-

transfected

-LRB-

C

-RRB-

transfected

-LRB-

D

-RRB-

and

-LRB-

-RRB-

doubly

transfected

and

-LRB-

E

-RRB-

etoposide

-LRB-

-RRB-

treated

-LRB-

Y-axis

cell

number

X-axis

DNA

content

-RRB-

Histograms

summarizing

flow

cytometric

analyses

of

transfected

cultures

-LRB-

F

-RRB-

apoptotic

and

-LRB-

G

-RRB-

apoptotic

Daoy

in

with

plasmids

and

treatment

with

as

indicated

-LRB-

Y-axis

X-axis

transfected

plasmid

-LRB-

s

-RRB-

and

culture

conditions

-RRB-

The

following

annotation

symbols

refer

to

statistically

significant

differences

in

cell

populations

as

determined

by

paired

Student

t-test

cells

transfected

with

plasmid

versus

mock-transfected

cells

-LRB-

P

-RRB-

cells

co-transfected

with

both

and

plasmids

versus

cells

transfected

with

alone

-LRB-

P

-RRB-

cells

transfected

with

plasmid

and

treated

with

vs.

mock-transfected

cells

-LRB-

P

-RRB-

cells

transfected

with

plasmid

versus

untreated

max-GFP

control-transfected

cells

-LRB-

P

-RRB-

Daoy

cells

transfected

with

plasmid

and

treated

with

vs.

mock-transfected

Daoy

cells

-LRB-

P

-RRB-

Increasing

in

cells

with

a

GFP-tagged

expression

plasmid

also

increased

apoptosis

-LRB-

in

-RRB-

compared

to

mock-transfected

controls

-LRB-

P

-RRB-

-LRB-

Fig.

-RRB-

treatment

of

cells

further

increased

the

population

to

-LRB-

P

compared

to

mock-transfected

controls

-RRB-

-LRB-

Fig.

-RRB-

These

results

suggest

that

additional

constitutive

expression

in

transfected

cells

can

overcome

negative

regulatory

pathways

including

endogenous

and

We

also

transfected

the

Daoy

medulloblastoma

cell

line

Like

cells

control

Daoy

cells

treated

with

increased

their

apoptotic

population

to

vs.

in

untreated

controls

suggesting

that

induces

apoptosis

even

in

the

absence

of

wild-type

-LRB-

Fig.

-RRB-

Constitutive

expression

of

transfected

wild-type

in

Daoy

cells

enhanced

apoptosis

to

-LRB-

-RRB-

compared

to

mock-transfected

controls

-LRB-

P

-RRB-

-LRB-

Fig.

-RRB-

These

data

suggest

that

transfected

expression

partially

rescues

mutation

in

Daoy

cells

overexpression

limits

and

apoptosis

We

examined

the

effects

of

constitutive

expression

in

cells

exposure

increased

apoptosis

to

from

in

untreated

-LRB-

Fig.

-RRB-

Despite

limited

transfection

efficiency

extra

expression

appeared

to

antagonize

activity

even

in

the

absence

of

genotoxic

stress

When

cells

were

co-transfected

to

constitutively

overexpress

both

and

they

displayed

fewer

cells

in

than

-LRB-

vs.

respectively

P

-RRB-

-LRB-

Fig.

-RRB-

These

data

suggest

that

additional

limits

exogenous

apoptosis

In

double

or

cells

increased

apoptosis

-LRB-

-RRB-

-LRB-

Fig.

-RRB-

Compared

to

double

transfectants

displayed

a

trend

toward

less

apoptosis

and

fewer

cells

in

phase

-LRB-

Fig.

-RRB-

These

data

are

consistent

with

apoptosis

presumably

due

to

overexpression

despite

genotoxic

stimulation

Daoy

cells

lacking

wild-type

did

not

differ

in

their

cell

cycle

profile

from

control

transfectants

under

basal

conditions

demonstrating

the

of

effects

However

underwent

more

apoptosis

-LRB-

-RRB-

than

untreated

counterparts

-LRB-

-RRB-

-LRB-

Fig.

-RRB-

These

data

suggest

a

minimal

effect

of

constitutively

expressed

in

the

absence

of

wild-type

Overexpressed

also

limited

apoptosis

in

doubly

co-transfected

Daoy

cells

As

in

controls

treatment

of

double

Daoy

transfectants

increased

cells

in

-LRB-

-RRB-

-LRB-

Fig.

-RRB-

However

double

or

displayed

fewer

apoptotic

cells

compared

to

single

Thus

excess

also

appears

to

protect

Daoy

cells

from

arrest

and

apoptosis

Discussion

Regulation

of

the

responses

of

medulloblastoma

to

genotoxic

therapies

is

not

well

understood

Because

has

been

implicated

but

rarely

mutated

or

deleted

in

medulloblastoma

we

focused

attention

on

the

candidate

oncogene

Recently

published

studies

employed

genomic

methods

to

associate

with

gain

of

in

medulloblastomas

Mendrzyk

et

al.

used

array-CGH

to

identify

gain

of

chromosomal

region

a

locus

containing

in

of

-LRB-

-RRB-

medulloblastomas

-LSB-

-RSB-

mRNA

was

moderately

overexpressed

with

respect

to

normal

cerebellum

control

in

seven

of

nine

tumors

Immunostaining

of

a

large

series

of

tissue

micro-arrays

revealed

strong

nuclear

expression

in

of

-LRB-

-RRB-

tumor

samples

Ehrbrecht

et

al.

used

conventional

and

array-CGH

to

identify

amplification

of

in

three

-LRB-

-RRB-

tumors

and

copy

number

gain

of

in

another

out

of

medulloblastomas

-LSB-

-RSB-

In

of

-LRB-

-RRB-

tumors

mRNA

was

expressed

less

than

fivefold

in

excess

of

expression

in

normal

cerebellum

control

Using

additional

approaches

we

have

confirmed

gain

of

the

locus

in

medulloblastomas

and

quantitated

its

relative

overexpression

in

our

larger

series

of

tumors

These

results

raise

the

possibility

that

overexpressed

functions

as

an

oncoprotein

in

medulloblastoma

is

a

gene

induced

by

genotoxic

stress

-LSB-

-RSB-

As

a

serine

threonine

phosphatase

family

member

can

counteract

the

tumor

suppressive

activity

of

through

several

distinct

mechanisms

dephosphorylates

that

has

been

activated

in

response

to

genotoxic

stress

-LSB-

-RSB-

also

dephosphorylates

and

stabilizes

promoting

degradation

-LRB-

Xiongbin

Lu

manuscript

submitted

-RRB-

This

provides

another

feedback

loop

that

returns

the

cell

to

a

homeostatic

state

following

DNA

repair

can

complement

different

oncogenes

in

their

transformation

of

mouse

embryo

fibroblasts

overexpression

also

phenocopies

the

anti-apoptotic

effects

of

mutation

in

vitro

and

increases

tumorgenicity

in

murine

models

in

vivo

-LSB-

-RSB-

Amplification

or

overexpression

of

in

breast

and

ovarian

cancers

may

phenocopy

the

loss

of

function

thereby

contributing

to

growth

and

resistance

to

genotoxic

therapies

-LSB-

-RSB-

In

breast

carcinomas

with

amplified

and

overexpressed

the

locus

is

rarely

mutated

suggesting

that

amplification

inhibits

activity

and

reduces

selection

for

mutations

during

tumor

progression

-LSB-

-RSB-

Although

expression

reportedly

correlates

with

clinical

outcomes

in

patients

with

breast

and

ovarian

carcinomas

-LSB-

-RSB-

conclusive

multivariate

analysis

of

additional

factors

-LRB-

such

as

the

concurrent

deletion

of

a

allele

and

loss

of

seen

with

-RRB-

awaits

future

analysis

of

greater

tumor

numbers

Analysis

of

our

series

of

primary

medulloblastomas

revealed

low-level

amplification

of

as

well

as

increased

expression

of

mRNA

transcript

and

protein

especially

in

tumors

with

gain

and

The

connection

between

amplification

and

these

common

cytogenetic

lesions

suggests

a

selective

advantage

Since

a

few

tumors

with

did

not

display

amplification

other

factors

probably

contribute

to

the

emergence

of

this

characteristic

cytogenetic

lesion

Conversely

one

or

more

tumors

without

gain

displayed

overexpression

Although

extra

copy

numbers

may

explain

relative

overexpression

via

gene

dosage

effects

regulation

at

the

post-transcriptional

and

translational

levels

may

also

affect

protein

levels

Consistent

with

our

FISH

results

qRT-RTPCR

and

Western

blot

analyses

demonstrated

increased

levels

of

mRNA

and

protein

both

significantly

associated

with

gain

and

The

degree

of

RNA

overexpression

relative

to

normal

cerebellar

tissue

corresponded

to

earlier

reports

-LSB-

-RSB-

The

apparent

discrepancy

between

expression

at

the

RNA

and

protein

levels

suggests

that

post-translational

modifications

also

contribute

to

overexpression

The

positive

and

negative

regulatory

networks

appear

to

involve

and

other

DNA

damage

response

factors

-LSB-

-RSB-

Of

note

our

analysis

was

limited

to

those

tumor

specimens

with

available

fresh

tissue

for

molecular

studies

and

displays

an

unintended

selection

bias

for

mixed

tumors

with

anaplastic

features

However

of

our

findings

appeared

to

correlate

with

histologic

subtype

or

clinical

features

Having

confirmed

the

gain

of

copy

numbers

and

quantitated

its

relative

overexpression

in

our

medulloblastoma

series

we

hypothesized

that

overexpressed

contributes

to

treatment

resistance

by

interfering

with

function

in

medulloblastoma

Because

the

tumor

specimens

were

obtained

before

genotoxic

treatment

their

and

levels

do

not

reflect

their

activity

Therefore

we

employed

established

cell

lines

to

analyze

the

effects

of

overexpression

of

Despite

their

phenotypic

differences

and

Daoy

cells

provide

readily

manipulable

systems

for

comparing

the

cell

cycle

effects

of

in

the

setting

of

wild-type

versus

mutant

Our

results

indicate

that

chemotherapy-induced

genotoxic

stress

or

overexpression

alone

can

induce

apoptosis

in

cells

The

constitutive

overexpression

of

overcame

endogenous

negative

regulation

in

cells

and

rescued

in

Daoy

cells

These

results

are

consistent

with

studies

of

other

wild-type

cell

types

including

the

neuroepithelial

lineage

indicating

that

induces

apoptosis

in

a

manner

-LSB-

-RSB-

Medulloblastoma

cell

lines

displayed

predicted

effects

of

overexpression

In

addition

to

high

basal

levels

in

cells

additional

transfected

counteracted

the

apoptotic

effects

of

endogenous

The

and

chemoprotective

effects

of

overexpressed

were

also

evident

upon

treatment

These

results

support

the

hypothesis

that

overexpression

modulates

genotoxic

responsiveness

by

negatively

regulating

in

medulloblastoma

cells

overexpression

appears

to

antagonize

activation

and

provides

medulloblastoma

cells

the

ability

to

resist

arrest

and

apoptosis

due

to

genotoxic

stress

The

frequency

of

amplification

and

the

degree

of

overexpression

in

primary

medulloblastoma

strongly

suggests

the

acquisition

of

selective

advantages

that

may

explain

the

prevalence

of

gain

and

Based

on

evidence

from

medulloblastoma

cell

lines

we

hypothesize

that

regulates

function

which

contributes

to

tumor

growth

and

genotoxic

treatment

resistance

The

recent

development

and

characterization

of

agents

that

inhibit

raise

the

possibility

of

therapeutic

intervention

in

those

medulloblastomas

with

wild-type

-LSB-

-RSB-

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

medulloblastoma
p53
isochromosome 17q
fish
apoptosis