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DNA methylation markers for blood-based detection of small cell lung cancer in mouse models
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DNA methylation markers for blood-based detection of small cell lung cancer in mouse models
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Content
1
DNA
Methylation
Markers
for
Blood-‐Based
Detection
of
Small
Cell
Lung
Cancer
in
Mouse
Models
by
Honey
Alef-‐Omidy
________________________________________________
A
Thesis
Presented
to
the
FACULTY
OF
THE
USC
GRADUATE
SCHOOL
UNIVERSITY
OF
SOUTHERN
CALIFORNIA
In
Partial
Fulfillment
of
the
Requirements
for
the
Degree
MASTER
OF
SCIENCE
(Biochemistry
and
Molecular
Biology)
Principal
Investigator:
Ite
A.
Laird-‐Offringa,
Ph.D.
December
2014
Copyright
2014
Honey
Alef-‐Omidy
2
Acknowledgement
I
would
like
to
first
thank
my
Principal
Investigator,
Dr.
Ite
Laird-‐Offringa
for
her
help
and
support.
She
has
been
so
generous
with
her
time
and
knowledge
since
the
first
day
I
started
working
in
her
research
laboratory.
I
would
like
to
thank
her
for
giving
me
a
chance
to
explore
and
understand
the
theories
and
practice
of
biochemistry
and
molecular
biology.
She
has
given
me
the
opportunity
to
further
employ
my
theoretical
knowledge
by
putting
it
in
practice
during
the
assignment
of
my
project.
Through
her
classes,
meetings
and
suggestions
I
was
able
to
further
expand
my
knowledge
and
love
for
cancer
biology,
biochemistry
and
genetics.
Dr.
Ite
Laird-‐Offringa
is
a
great
mentor,
professor
and
friend
who
always
cares
about
the
well-‐being
of
her
students.
I
would
also
like
to
thank
Dr.
Mihaela
Campan,
MD.
Ph.D.,
Mario
Pulido
and
Evelyn
Tran,
for
all
their
help
and
dedication
in
the
Laird-‐Offringa
lab.
They
were
always
there
to
teach
and
guide
me
with
my
project.
Their
enthusiasm
for
research
gave
me
the
motivation
to
continue
working
on
my
project.
I
would
also
like
to
thank
my
committee
members,
Dr.
Zoltan
Tokes
and
Dr.
Daniel
Weisenberger
for
their
time.
I
would
like
to
thank
them
both
for
being
great
professors.
They
have
influenced
my
learning
experience,
and
scientific
knowledge
and
for
that
I
am
very
grateful.
I
would
also
like
to
thank
the
members
of
the
Laird-‐Ofrringa
lab
for
making
the
lab
setting
a
pleasant
experience.
Their
help
and
support
made
the
course
of
my
project
an
easy
and
enjoyable
time.
Also,
I
would
like
to
thank
the
Department
of
Biochemistry
and
Molecular
Biology
faculty
and
staff
for
giving
me
the
opportunity
to
learn
and
discover
my
love
for
3
science.
The
University
of
Southern
California
will
always
be
in
my
heart
wherever
I
go
and
I
will
always
be
proud
to
say
that
the
Biochemistry
and
Molecular
Biology
department
shaped
my
scientific
knowledge.
My
motivation
to
attend
graduate
school
was
my
curiosity
to
learn
more
about
the
fields
of
cancer
biology,
genetics
and
biochemistry
and
molecular
biology
and
then
apply
the
knowledge
to
make
significant
contributions
through
my
research.
My
plans
after
graduation
are
to
work
hard
for
my
future
goal,
which
is
to
sign
up
to
a
Ph.D.
program.
This
will
allow
me
to
expand
my
knowledge
in
science,
a
field
that
I
profoundly
love.
Finally,
I
would
also
like
to
thank
my
family,
my
lovely
parents
and
brothers
in
Iran,
as
well
as
my
sweet
relatives
in
the
U.S.
for
their
love
and
support.
I
thank
my
parents
for
all
the
love
and
encouragement
throughout
my
scholastic
experience.
They
have
always
supported
me
in
everything
I
wanted
to
accomplish
in
life
4
Table
of
Contents
Acknowledgement……………………….……....………..………………………………….…………………………………….2
List
of
Figures……………………………………………………………..……………………………….…………………..……....6
List
of
Tables………………………………………………………………………………………………..…………………..……....7
Abstract………………………………………………………………………………………………….………………………………...8
Chapter
One:
Introduction…………...………..………………………………………….…………………………….…….10
1-‐A.
Epigenetic
modifications
leading
to
cancer
developments………………….………….….….……10
1-‐B.
Lung
Cancer
and
its
most
aggressive
subtype
(SCLC)……………….….…………………….…..……16
1-‐C.
Animal
models
as
tools
for
understanding
the
SCLC:
The
mouse
as
a
popular
model
in
the
study
of
SCLC
system…….…………..……….…………………………....……………………….…………..……19
1-‐D.
Using
DNA
methylation
markers
for
SCLC
detection……….……………..……………………….….23
Chapter
Two:
Materials
and
Methods…….……………………….……………………………………..………………27
2.1.
Tumor
induction
in
mice,
mouse
dissection,
tissue
collection……………………………....27
2.1-‐A.
Tumor
induction
in
the
SCLC
mouse
model………………………………………..………..27
2.1-‐B.
Mouse
tissue
collection…………………………………………………...…………………….30
2.2.
DNA
Extraction
from
tissue
samples………………….………………….…………..………………….31
2.2-‐A.
DNA
extraction
from
the
tissues
preserved
in
OCT
or
those
directly
frozen…………………….…………………………………………………..…………………………………...…….31
2.2-‐A-‐I.
Tissue
Lysis……………………………………………….………………………………………….31
5
2.2-‐A-‐II.
Phenol-‐
Chloroform
extraction……………………….…………………..……...………32
2.2-‐B.
DNA
extraction
from
paraffin-‐embedded
tissues……..……………….………..……….34
2.3.
In
vitro
methylation
of
normal
mouse
DNA......…….……………………….……..………....……35
2.4.
Bisulfite
Conversion.…………….…………..……………………………………………........................35
2.5.
MethyLight
analysis……….……………………………………………..……………………………….….….38
Chapter
Three:
Results………………..………….………………………………………………………………………..…....41
3-‐A.
SCLC
induction
in
FVB/N
mice
and
tissue
specimen
collection
from
induced
and
un-‐
induced
FVB/N
mice
and
C57
mice.........................................................................................41
3-‐B.
In
vitro
DNA
methylation
(M.SssI
treatment)
Results……………………….………….……..…….43
3-‐C.
MethyLight
analysis
Results
for
microdissected
SCLC
tumors………..…………….…….……....47
3-‐D.
MethyLight
analysis
Results
for
SCLC
tumors
that
were
not
microdisected...................48
Chapter
Four:
Discussion………..…………………………..…………….…………………………………………….….….50
References/Bibliography………………...……………………………………….………………..………………………….53
6
List
of
Figures
Figure
Number/Name
page
1.
DNA
methylation.……….…………………………………………………………………………..……………………...…..11
2.
How
DNA
methylation
can
have
effect
on
target
gene
expression
in
normal
cells….……….…..12
3.
DNA
modifications
in
cancer.
…………………..…………………………………………………………………….…..14
4.
Hypo
and
hyper
methylation
at
CpG
islands
leading
to
tumorogenesis….……………………..……..15
5.
What
are
the
Types
of
Lung
Cancer?
.……………………………………………………………..………..…………16
6.
Schematic
picture
of
different
lung
cell
types……………………………………………………………………...18
7.
Illustration
of
the
design
of
the
Dr.
Anton
Bern’s
and
his
colleagues
SCLC
model……..…..…….22
8.
DNA
Methylation
as
a
biomarker
for
early
detection
of
lung
cancer.…………………………….……..23
9.
Bisulfite
Conversion………..……………………………………………………………………………………………………25
10.
Sodium
bisulfite
modifies
the
sequences
of
genomic
DNA.………………………………….…...……….26
11.
Types
of
reactions
used
in
the
MethyLight
assay…..……………………………………..………..………….26
12.
Tracheal
intubation
and
injection
of
Adeno-‐Cre
virus
in
the
mouse.………………………….………29
13.
Two
SCLC
tumor
tissue
specimens
from
FVB/N
induced
mouse
in
melted
OCT………………...32
14.
Phase
separation
in
Phenol-‐chloroform
extraction….….……………………………………………….……32
15.
Collecting
DNA
pellet
from
phenol/Chloroform
DNA
extract
with
centrifuge….…………………33
16.
Paraffin
embedded
SCLC
tissue
samples………………..……………………………………………………...….34
17.
Conversion
of
non-‐methylated
cytosine
by
DNA
Bisulfite
conversion
…………………………..……37
18.
Schematic
procedure
of
the
Quantitative
PCR…………………………………………………………………...48
19.
PMR
(Percent
of
Methylated
Reference)
calculation……………………………….…………………….…..39
20.
Evaluation
DNA
methylation
levels
by
ΔCt
determination.…………………………………………………44
7
List
of
Tables
Table
Number/Name
Page
1.
Summary
of
the
tissue
samples
used
in
the
MethyLight
analysis..……….………………………………43
2.
FVB/N-‐
M-‐SssI
first
Treatment
results……….……………….………………………………..………….……..……46
3.
FVB/N-‐
M-‐SssI
second
Treatment
results.……………………………………..…………….…..……….…………46
4.
C57Bl/6-‐
M-‐SssI
Treatment,
First
determination..……………………………………………………………..…47
5.
C57Bl/6-‐
M-‐SssI
Treatment,
second
determination..……………………….…….…………………….………47
6.
PMR
values
from
paraffin
embedded
tissues
(Micodissected)…………………………………..…………48
7.
PMR
valuess
from
MethyLight
reaction
on
7
SCLC
tumors
DNA
and
one
normal
lung
DNA….49
8.
DNA
methylation
differences
in
frozen
tissues
and
fresh
frozen
paraffin
embedded……….….50
8
Abstract
Lung
cancer
is
the
top
cancer
killer
in
the
United
States.
This
cancer
starts
in
the
cells
lining
the
bronchi
or
other
parts
of
the
lung
such
as
bronchioles
or
alveoli.
It
can
be
classified
into
two
major
groups:
small
cell
lung
cancer
(SCLC)
and
non-‐small
cell
lung
cancer
(NSCLC).
Although
SCLC
is
less
common
than
NSCLC
it
is
the
most
aggressive
type
of
lung
cancer.
To
this
day,
there
are
no
effective
serum
biomarkers
for
detection,
prediction
of
response
or
monitoring
this
disease.
Such
markers
could
be
very
valuable
because
currently
most
SCLC
patients
are
diagnosed
after
their
cancer
has
metastasized,
and
there
is
no
way
apart
from
imaging
to
test
for
response
or
recurrence.
It
has
been
amply
demonstrated
that
epigenetic
modifications,
such
as
alterations
in
cytosine-‐5
DNA
methylation,
occur
frequently
in
all
types
of
cancer.
DNA
methylation
can
be
detected
in
the
serum
of
cancer
patients
using
a
sensitive
assay
called
MethyLight.
Our
lab
has
developed
blood-‐based
DNA
methylation
markers
for
NSCLC
that
have
also
been
shown
to
be
present
in
the
blood
of
small
cell
lung
cancer
patients.
We
were
interested
to
determine
whether
these
markers
are
also
present
in
tumors
derived
from
a
mouse
model
of
SCLC.
These
mice
are
important
tools
to
develop
new
therapies,
and
identify
molecular
markers
that
could
be
used
to
determine
whether
the
mice
are
responding
to
treatment
or
showing
recurrent
disease
would
be
very
useful.
We
extracted
the
DNA
from
normal
and
tumor
tissue
from
mice
with
SCLC
and
disease-‐free
control
mice.
Prior
to
DNA
extraction,
tissues
were
either
microdissected
from
paraffin
blocks
or
were
directly
taken
from
a
frozen
specimen.
We
performed
MethyLight
analysis
on
these
DNAs
and
compared
the
methylation
levels
between
normal
lung
and
tumors.
In
order
to
setup
9
the
MethyLight
assay
for
mouse
DNA,
we
required
a
(positive
control)
fully
in
vitro
methylated
DNA
sample
to
serve
as
reference
in
the
MethyLight
assay.
We
generated
large
amounts
(3-‐
5μg)
of
in
vitro
methylated
DNA
for
two
different
strains
of
mice.
This
DNA
is
an
important
resource
for
the
lab
since
it
can
be
used
as
a
positive
control
MethyLight
assays
for
years
to
come,
and
eliminates
the
biases
associated
with
using
different
batches
of
reference
DNA.
MethyLight
analysis
of
normal/tumor
DNA
from
the
SCLC
mouse
model
showed
increased
methylation
in
some
of
the
tumors
when
the
microdissected
material
was
used,
but
little
or
no
methylation
when
the
frozen
samples
were
examined.
These
results
suggest
that
microdissection
might
be
important
in
DNA
methylation
analysis.
Because
modest
levels
of
methylation
were
detected
but
no
marker
showed
high
sensitivity,
we
suggest
that
a
genome-‐
wide
analysis
of
DNA
methylation
patterns
in
these
mice
would
be
useful.
It
might
uncover
DNA
methylation
abnormalities
that
are
specific
for
mice
with
SCLC.
Key
Words:
Small
cell
Lung
Cancer,
DNA
Methylation,
Biomarkers,
Mouse
models,
MethyLight,
Sodium
bisulfite.
10
Chapter
one
Introduction
1-‐A.
Epigenetic
modifications
leading
to
cancer
development
Epigenetics
has
been
defined
as
the
study
of
the
mitotically
and/or
meiotically
heritable
information
that
is
layered
on
top
of
the
genome,
affecting
the
control
of
gene
expression
without
changing
the
DNA
sequence.
[1]
Epigenetic
modifications
play
an
important
role
in
normal
development
and
maintenance
of
tissue-‐specific
gene
expression
patterns
in
mammals.
Abnormal
epigenetic
modifications
have
been
also
shown
to
contribute
to
common
human
diseases
such
as
cancer.
The
ability
of
genes
to
alter
their
expression
is
controlled
by
epigenetic
factors
such
as
DNA
methylation.
[2],
[3]
DNA
methylation
is
a
chemical
modification
of
DNA
in
which
a
methyl
group
(CH3)
is
added
to
DNA
nucleotides.
In
mammals
the
methyl
group
largely
is
added
to
the
C5
position
of
the
cytosine
pyrimidine
ring
[4]
(Figure
1).
DNA
methylation
was
discovered
before
the
structure
of
DNA
was
resolved
[5]
DNA
methylation
occurs
at
small
inverted
repeats
called
CpG
dinucleotides
(cytosine
(C)
next
to
a
guanine
(G)
nucleotide
in
the
linear
sequence
of
bases
separated
by
a
phosphate
link
(p)).
The
"CpG"
notation
is
used
to
distinguish
this
linear
sequence
from
the
CG
base-‐pairing
of
cytosine
11
and
guanine
[6].
Enzymes
that
add
the
methyl
group
are
called
DNA
methyltransferases
(DNMTs)
(Figure
1)
Figure
1.
DNA
methylation:
Methylation
of
cytosines
at
their
5
th
carbon
position
with
the
help
of
DNMT
enzyme
[7]
(Zakhari
S:
Alcohol
metabolism
and
epigenetics
changes.
Alcohol
Res.
2013;
35(1):
6-‐16.)
Normally,
between
60%
and
90%
of
all
CpGs
are
methylated
in
mammals.
[8][9]
Over
time
methylated
cytosine
residues
can
spontaneously
deaminate
to
form
thymine
(T)
residues;
hence,
in
nature
CpG
dinucleotides
that
are
normally
methylated
have
been
steadily
depleted,
which
is
evidenced
by
the
under-‐representation
of
CpG
dinucleotides
in
the
human
genome
except
in
those
regions
that
are
not
methylated.
[10]
12
DNA
methylation
fulfills
the
following
functions
in
humans
[11]:
(1.)
Regulation
of
gene
expression:
In
order
to
maintain
proper
functioning
of
cellular
processes,
gene
expression
must
be
tightly
controlled.
Short
term
changes
in
expression
patterns
are
usually
induced
by
transcription
factors,
whereas
long-‐term
gene
expression
regulation
is
achieved
by
a
complex
interplay
of
chromatin
remodeling,
modifications
of
DNA-‐bound
histones,
nuclear
positioning
of
chromosomal
regions,
regulatory
ribonucleic
acids
(RNAs)
and,
last
but
not
least,
DNA
methylation
[12-‐14].
DNA
methylation
of
gene
promoters
is
usually
associated
with
absence
of
gene
expression
(Figure
2).
Figure
2.
How
DNA
methylation
can
have
effect
on
target
gene
expression
in
normal
cells.
(Image:
James
D.Fry,
University
of
Rochester,
2011)
13
(2.)
Genomic
imprinting:
Genomic
imprinting
refers
to
genomic
regions
from
which
only
the
paternal
or
the
maternal
allele
is
expressed.
This
parent-‐of-‐origin-‐specific
expression
pattern
is
mediated
by
epigenetic
modifications,
including
DNA
methylation,
of
either
the
paternal
or
maternal
allele
[15].
(3.)
X-‐chromosomal
inactivation:
DNA
methylation
is
involved
in
the
inactivation
of
one
of
the
two
X-‐chromosome
copies
present
in
female
cells.
This
process
ensures
that
similar
amounts
of
X-‐chromosomal
gene
products
are
expressed
in
both
males
and
females
[16].
(4.)
Genome
defense:
Parasitic
genomic
elements
like
retrotransposons
are
inactivated
by
DNA
methylation,
making
DNA
methylation
a
key
player
in
the
maintenance
of
genome
integrity
[17].
The
unmethylated
CpGs
are
often
grouped
in
clusters
called
CpG
islands,
which
are
often
present
in
the
5'
regulatory
regions
(promoters)
of
genes.
In
many
disease
processes,
such
as
cancer,
promoter
CpG
islands
acquire
abnormal
hypermethylation,
which
may
results
in
transcriptional
silencing
that
can
be
inherited
by
daughter
cells
following
cell
division.
(Figure
3)
14
Figure
3.
DNA
modifications
in
cancer.
[18]
One
of
the
DNA
methylation
Functions
is
regulating
the
gene
expression.
As
it
is
shown
in
this
figure
promoter
CpG
Islands
are
unmethylated
in
normal
cells,
while
they
have
acquired
abberant
DNA
methylation
following
transcriptional
silencing
in
cancers
cells.
Thus
alteration
of
DNA
methylation
has
been
known
as
an
important
component
for
cancer
development.
(Image
obtained
from:
Epigenetic
and
Cancer
review,
Q.W.
Chen
et
al.,
2013)
Alterations
of
DNA
methylation
have
been
recognized
as
an
important
component
of
cancer
development.DNA
Hypomethylation,
in
general,
arises
earlier
and
is
linked
to
chromosomal
instability,
loss
of
imprinting,
or
over-‐expression
of
oncogenes
within
cancer
cells
(Figure
4
[19]).
Global
hypomethylation
has
also
been
implicated
in
the
development
and
progression
of
cancer
through
different
mechanisms
(Figure
4
[20]).
Hypermethylation
typically
occurs
at
CpG
islands
in
the
promoter
region
and
is
associated
with
gene
inactivation
(Figure
3).
Methylation
of
CpG
sites
within
the
promoters
of
genes
can
lead
to
their
silencing,
for
example
of
tumor
suppressor
genes,
a
feature
found
in
a
numerous
human
cancers
(Figure
4).
15
Figure
4.
Hypo
and
hyper
methylation
at
CpG
islands
leading
to
tumorogenesis.
(Image
obtained
from:
DNA
methylation
and
human
disease,
Keith
D,
Robertson,
Nature
review,2005)
The
diagram
shows
a
representative
region
of
genomic
DNA
in
a
normal
cell.
The
region
shown
contains
repeat-‐
rich,
hypermethylated
pericentromeric
heterochromatin
and
an
actively
transcribed
tumor
suppressor
gene
(TSG)
associated
with
a
hypomethylated
CpG
island
(indicated
in
red).
In
tumor
cells,
repeat-‐rich
heterochromatin
becomes
hypomethylated
and
this
contributes
to
genomic
instability,
a
hallmark
of
tumor
cells,
through
increased
mitotic
recombination
events.
De
novo
methylation
of
CpG
islands
also
occurs
in
cancer
cells,
and
can
result
in
the
transcriptional
silencing
of
growth-‐regulatory
genes.
These
changes
in
methylation
can
be
early
events
in
tumorigenesis.
[21]
DNA
methylation
may
affect
the
transcription
of
genes
in
two
ways.
First,
the
methylation
of
DNA
itself
may
physically
impede
the
binding
of
transcription
factors
to
a
gene
or
regulatory
element
[22].
Second,
and
likely
more
important,
methylated
DNA
may
be
bound
by
proteins
known
as
methyl-‐CpG-‐binding
domain
proteins
(MBDs).
MBD
proteins
then
recruit
additional
proteins
to
the
locus,
such
as
histone
deacetylases
and
other
chromatin
remodeling
proteins
that
can
modify
histones,
thereby
forming
compact,
inactive
chromatin,
termed
heterochromatin.
This
link
between
DNA
methylation
and
chromatin
structure
is
very
16
important.
In
particular,
loss
of
methyl-‐CpG-‐binding
protein
2
(MeCP2)
has
been
implicated
in
Rett
syndrome;
and
methyl-‐CpG-‐binding
domain
Protein
2
(MBD2)
mediates
the
transcriptional
silencing
of
hyper-‐methylated
genes
in
cancer.
[6]
In
the
clinical
setting,
DNA
methylation
is
an
exciting
therapeutic
target
for
cancer
treatment;
methylation
inhibiting
agents
are
now
routinely
used
in
the
treatment
of
several
malignancies.
[24]
1-‐B.
Lung
Cancer
and
its
most
aggressive
subtype
(SCLC)
Lung
cancer
is
the
top
cancer
killer
in
both
men
and
women
around
the
world.
Based
on
the
American
Cancer
Society
report,
an
estimated
159,260
Americans
are
expected
to
die
from
lung
cancer
in
2014
[56].
Lung
cancer
death
accounts
for
approximately
27%
of
all
cancer
deaths
There
are
two
main
types
of
lung
cancer
(Figure
5):
Figure
5.
What
are
the
Types
of
Lung
Cancer?
(By:
American
Lung
Association)[57]
17
Non-‐small
cell
lung
cancer
(NSCLC)
Non-‐small
cell
lung
cancer
is
the
most
common
type
of
lung
cancer.
It
makes
up
about
80
percent
of
all
lung
cancer
cases.
It
is
subdivided
into
three
major
subtypes:
lung
adenocarcinoma,
squamous
cell
carcinoma,
and
large
cell
carcinoma.
In
addition,
carcinoids
and
other
minor
types
make
up
about
5%
of
NSCLCs.
Small
cell
lung
cancer
(SCLC)
represents
10%
to
15%
of
all
lung
cancers.
It’s
named
for
the
size
of
the
cancer
cells
when
seen
under
a
microscope.
Other
names
for
SCLC
are
oat
cell
cancer,
oat
cell
carcinoma,
and
small
cell
undifferentiated
carcinoma.
SCLC
often
starts
in
the
bronchi
near
the
center
of
the
chest.
It
tends
to
grow
and
spread
more
quickly
than
non-‐small
cell
lung
cancer.
It
has
been
shown
that
SCLC
has
a
more
rapid
doubling
time,
a
higher
growth
fraction,
and
earlier
development
of
widespread
metastases.
Despite
of
a
more
dramatic
initial
response
to
chemotherapy
and
radiation
therapy,
most
patients
die
due
to
recurring
disease
[23].
Untreated
SCLC
has
the
most
aggressive
clinical
course
of
any
lung
tumor,
with
a
median
survival
of
only
2
to
4
months
after
diagnosis.
[24].
Survival
statistics
of
lung
cancer
are
grim
because
of
its
late
detection
and
frequent
local
and
distal
metastases.
Although
DNA
sequence
information
from
tumors
has
revealed
a
number
of
frequently
occurring
mutations,
affecting
well-‐known
tumor
suppressor
genes
(such
as
loss
of
function
in
Rb1
and
P53
genes)
and
proto-‐oncogenes
(gain
of
function
or
over
expression),
many
of
the
driver
mutations
remain
to
be
defined.
This
is
likely
due
to
the
involvement
of
18
numerous
rather
infrequently
occurring
driver
mutations
that
are
difficult
to
distinguish
from
the
very
large
number
of
passenger
mutations
detected
in
smoking-‐related
lung
cancers
[25].
The
low
survival
of
patients
with
SCLC
(5-‐year
survival
of
6%)
points
to
the
necessity
of
finding
means
of
detecting
these
cancers
at
early
stages
at
which
point
in
time
the
surgical
resection
of
the
tumor
could
extend
the
life
of
SCLC
patients.
Lung
cancers
start
in
the
cells
lining
the
alveoli
and
the
bronchioles
as
well
as
other
cell
types
located
in
the
lung
such
as,
in
the
case
of
SCLC,
pulmonary
neuroendocrine
cells
[26].
Figure
6.
Schematic
picture
of
different
lung
cell
types
[27].
Most
SCLC
lesions
are
found
in
the
bronchioles
and
terminal
bronchioles,
majority
Clara
cells.
The
cell
of
origin
of
SCLC
has
not
been
formally
identified,
although
because
SCLC
expresses
neuroendocrine
(NE)
markers,
it
is
thought
to
arise
from
NECs
or
NEP.
NE
cells
are
located
in
the
lung
epithelium.
(Image:
Cell
of
Origin
of
lung
cancer,
Kate
D.
Sutherland
et
al.
2011)
19
The
cancer
may
take
time
to
grow.
During
which,
the
abnormal
cells
may
acquire
other
genetic
and
epigenetic
changes,
which
cause
them
to
progress
to
true
cancer.
As
a
cancer
develops,
the
cancer
cells
may
make
chemicals
that
cause
new
blood
vessels
to
form
nearby.
These
blood
vessels
nourish
the
cancer
cells,
which
can
continue
to
grow
and
form
a
tumor
large
enough
to
be
seen
on
imaging
tests
such
as
x-‐rays.
In
the
case
of
SCLC,
by
the
time
it
is
detectable
by
imaging
it
has
usually
already
metastasized
to
other
parts
of
the
body.
The
predominant
mutations
observed
in
human
SCLC
include
loss-‐of-‐function
mutations
in
the
retinoblastoma
(RB)
and
(Tp53)
genes.
Mutation
clustering
is
also
found
in
the
PI3K
pathway,
the
mediator
complex,
Notch
and
Hedgehog,
glutamate
receptors,
SOX
genes,
DNA
repair
genes
and
several
receptor
kinases
[28],
[29].
Gain
of
function
mutations
or
overexpression
of
proto-‐oncogenes
by
amplification
of
distinct
chromosomal
regions
includes
L-‐MYC,
C-‐MYC,
SOX2
and
SOX4
[29].
1-‐C.
Animal
models
as
tools
for
understanding
SCLC:
The
mouse
as
a
popular
model
in
the
study
of
SCLC
The
large
variety
of
lesions
found
in
SCLC
underscore
the
need
to
design
animal
models
in
which
the
importance
of
individual
lesions
can
be
assessed
in
the
context
of
various
combinations
of
concurrent
oncogenic
mutations.
This
notion
provides
a
strong
incentive
to
set-‐up
fast-‐track
mouse
tumor
models
in
which
different
combinations
of
lesions
can
be
evaluated
swiftly.
[30][31]
20
The
mouse
provides
a
good
model
to
study
human
development
and
disease
because
we
share
virtually
all
of
our
genes
and
use
them
in
similar
ways.
It
also
allows
the
investigators
to
perform
many
analyses
that
are
arduous
or
unachievable
to
carry
out
in
the
human
patients.
Using
mouse
models
to
study
diseases
such
as
cancer
has
numerous
advantages:
they
can
be
manipulated
and
treated
with
experimental
drugs,
in
addition
they
are
~
3
inches
long,
which
allows
us
to
keep
many
mice
in
a
room.
On
the
other
hand,
their
generation
time
is
about
3
months,
so
that
working
with
a
mouse
model
can
be
quite
time
consuming
depending
on
how
many
crosses
must
be
made.
It
is
also
expensive
to
maintain
a
mouse
colony.
Scientists
have
worked
with
mice
for
over
100
years
and
most
importantly
such
mice
have
been
used
with
genetic
tools
that
can
introduce
extra
genes
or
remove
a
specific
gene
allowing
studies
of
the
effect
of
mutations
on
development
and
cancer.
The
(RB)
and
(Tp53)
tumor
suppressor
genes
are
frequently
inactivated
in
SCLC
[32].
This
observation
has
been
the
basis
for
modeling
SCLC
in
compound
conditional
knockouts
of
retinoblastoma
(Rb1)
and
(Tp53)
genes
[33]. Using
a
Cre-‐loxP
system,
“
floxed”
Rb1
and
(Trp53)
were
homozygously
deleted
in
the
lung
epithelium
of
transgenic
mice
through
intra-‐tracheal
instillation
of
Adeno-‐Cre
virus.
All
treated
mice
develop
multiple
tumors
with
histopathology
and
immune-‐phenotype
similar
to
human
SCLC
beginning
around
200
days
post
infection
[34,35].
The
prolonged
lag
time
allows
for
the
monitoring
of
potential
immune
responses
against
various
SCLC-‐related
auto-‐antigens
prior
to
the
clinical
detection
of
the
disease.
It
can
take
more
than
6
months
before
tumors
become
evident.
The
tumors
closely
resemble
human
SCLC
and
even
metastasize
to
the
same
organs.
They
also
acquire
additional
mutations
that
are
21
reminiscent
of
human
SCLC,
such
as
the
amplification
of
one
of
the
Myc
genes
[36].
In
addition,
amplification
of
the
Nfib
gene
is
frequently
observed
in
mouse
SCLC
model.
This
gene
is
also
found
to
be
amplified
in
human
SCLC
[36].
The
prolonged
lag
time
for
tumor
development
allows
for
the
monitoring
of
potential
immune
responses
against
various
SCLC-‐related
auto
antigens
prior
to
the
clinical
detection
of
the
disease.
Recently,
it
was
observed
that
the
loss
of
p130,
a
cell
cycle
inhibitor
related
to
Rb1
[37]
that
normally
suppresses
SCLC
development
[38],
accelerates
the
development
of
SCLC
in
Rb1
/
Tp53
-‐mutant
mice.
Rb
/Tp53
/p130
mutant
mice
may
thus
provide
an
alternative
mouse
model
of
SCLC
with
a
shortened
lag
time
[39].
22
Figure
7.
Illustration
of
the
design
of
the
Dr.
Anton
Bern’s
and
his
colleagues
SCLC
model:
A:
Mice
that
carry
Rb
and
p53
genes
with
flanking
LoxP
sites
underwent
intratracheal
delivery
of
a
recombinant
adenoviral
vector
expressing
the
Cre
recombinase
(Ad-‐Cre),
which
cleaves
DNA
specifically
at
LoxP
sites.
In
those
mice
that
experienced
homozygous
loss
of
both
Rb1
and
p53,
SCLC
developed.[60]
B:
SCLC
metastases
were
detected
in
the
mouse
model
in
sites
commonly
involved
in
SCLC
patients.
While
the
frequencies
of
metastatic
involvement
in
the
mouse
model
were
not
described
[34][60],
the
percentages
of
newly
diagnosed
SCLC
patients
with
extra-‐thoracic
metastases
are,
by
site,
19%–38%
(bone),
17%–34%
(liver),
5%-‐31%
(adrenal
glands),
17%–23%
(bone
marrow),
0%–14%
(brain),
7%–25%
(lymph
nodes),
and
3%–11%
(soft
tissues)
[40]
(Image:
Minna
JD1,
Kurie
JM,
Jacks
T:
A
big
step
in
the
study
of
small
cell
lung
cancer.
Cancer
Cell.
2003
Sep;4(3):163-‐6.
PMID:
14522249.)
23
1-‐D.
Using
DNA
methylation
markers
for
SCLC
detection
Early
detection
of
SCLC
is
challenging
because
of
the
lack
of
adequate
biomarkers.
Therefore,
SCLC
is
usually
diagnosed
due
to
symptoms
associated
with
late
disease,
such
as
bulky
intra-‐
thoracic
malignancy
or
metastasis.
After
disease
is
suspected
due
to
symptoms,
other
invasive
tests
are
required
to
confirm
the
disease,
such
as
histological
analysis
of
bronchoscopic
samples
and
cytological
study
of
a
fine-‐needle
aspiration
(FNA),
endoscopic
ultrasound
(EUS)-‐guided
fine-‐needle
aspiration
(EUS-‐FNA),
or
transbronchoscopic
needle
aspiration
(TBNA)
Hence,
it
is
quite
important
to
identify
molecular
markers
that
might
help
with
the
detection
and
diagnosis
of
the
disease
that
can
ultimately
influence
the
patients’
survival.
Figure
8.
DNA
Methylation
as
a
biomarker
for
early
detection
of
lung
cancer.
This
model
proposes
that
as
the
number
of
genes
detected
in
sputum
from
current
or
former
smokers
increases,
the
relative
risk
for
lung
cancer
is
also
increased.
When
marker
multiplicity
reaches
a
certain
level
(this
is
being
assessed
through
case–
control
studies)
intervention
in
the
form
of
spiral
computed
tomography
and/or
bronchoscopy
would
be
recommended
to
identify
early
lung
cancer.
[41]
(Image:
Steven
A.
Belinsky:
Gene-‐promoter
hypermethylation
as
a
biomarker
in
lung
cancer.
Nat
Rev
Cancer.
2004
Sep;4(9):707-‐17.)
24
There
are
few
known
proteins
that
are
detectable
as
tumor
markers
in
serum
from
SCLC
patients
that
are
useful
as
putative
markers
of
the
disease.
These
proteins
have
a
neuroendocrine
origin
and
are
ubiquitously
present
in
neuroendocrine
tissues.
[42]
DNA
methylation
markers
have
recently
emerged
as
alternatives
to
protein
biomarkers
for
the
early
detection
of
cancer.
The
analysis
of
DNA
methylation
patterns
at
individual
gene
loci
has
traditionally
been
technically
challenging.
Two
main
techniques
have
been
employed:
methylation-‐sensitive
restriction
enzyme
digestion
and
sodium
bisulfite
conversion
of
DNA.
Methylation-‐sensitive
restriction
digestion
is
still
widely
employed
in
genome
wide
scanning
techniques
for
DNA
methylation
alterations,
such
as
restriction
landmark
genomic
scanning
(RLGS)
[43],
methylated
CpG
island
amplification
(MCA)
[44],
differential
methylation
hybridization
(DMH)
[45],
and
methylation-‐sensitive
arbitrarily
primed
PCR
(MS-‐AP-‐PCR)
[46].
Sodium
bisulfite
conversion
of
genomic
DNA
has
however
led
to
a
larger
number
of
new
methylation
analysis
techniques,
including
MethyLight,
MSP,
COBRA,
Q-‐MSPand
Ms-‐SNuPE.[47][50]
MethyLight
is
a
highly
sensitive
fluorescent-‐based
real-‐time
PCR
assay,
capable
of
detecting
methylated
alleles
in
the
presence
of
a
10,000-‐fold
excess
of
unmethylated
alleles.
The
assay
is
also
highly
quantitative
and
can
very
accurately
determine
the
methylation
levels
at
concordantly
methylated
CpGs
in
a
locus.
[47]
This
assay
allows
for
quantitative
DNA
methylation
analysis
at
a
specific
locus
by
using
DNA
oligonucleotides
that
anneal
differentially
to
bisulfite
converted
DNA
according
to
the
methylation
status
in
the
original
genomic
DNA.
25
[47][50]
Following
bisulfite
conversion,
methylated
cytosines
remain
unchanged,
while
unmethylated
cytosines
are
deaminated
to
uracils.
[48].
Changes
due
to
DNA
methylation
can
be
thus
converted
into
a
genetic
change
that
can
be
amplified
by
PCR
using
methylation-‐
specific
primers.
(Figure
9)
Figure
9.
Bisulfite
Conversion:
Scheme
illustrating
sodium
bisulfite-‐PCR
technique
for
identification
of
5-‐
methylcytosine
in
a
DNA
sequence.
5-‐mC,
C,
T,
A,
G,
U
indicate
5-‐methylcytosine,
cytosine,
thymine,
adenine,
guanine
and
uracil
respectively,
arrows
show
the
position
of
nitrogen
bases
on
the
DNA
strands
or
the
direction
of
the
reactions
[49]
(Image:
Mohammed
A.
Ibrahim:
Advances
in
Genomic
DNA
Methylation
Analysis.
Biotechnology
2010,
9:459468.DOI:10.3923/biotech.2010.459.468,URL:http://scialert.net/abstract/?doi=biotech.2010.459.468.)
The
high
sensitivity
and
specificity
of
MethyLight
make
it
uniquely
well
suited
for
detection
of
low-‐frequency
DNA
methylation
biomarkers
as
evidence
of
disease.
At
the
same
time,
the
quantitative
accuracy
of
real-‐time
PCR
and
the
flexibility
to
design
bisulfite-‐dependent,
methylation-‐independent
control
reactions
allows
for
a
quantitative
assessment
of
these
low-‐
frequency
methylation
events.
[48]
26
Figure
10.
Sodium
bisulfite
modifies
the
sequences
of
genomic
DNA
by
converting
unmethylated
Cs
(open
circles)
to
uracils
while
leaving
methylated
Cs
(solid
circles)
unmodified.
PCR
amplification
results
in
the
replacement
of
uracil
residues
by
thymines.
Note
that
bisulfite
conversion
destroys
the
self-‐complementarity
of
the
original
genomic
DNA,
so
that
two
different
PCR
products
can
be
generated:
one
derived
from
the
top
strand,
and
one
derived
from
the
bottom
strand.
[50][40[[51]
(Image:
Trinh
BN1,
Long
TI,
Laird
PW:
DNA
methylation
analysis
by
MethyLight
technology.
Methods.
2001
Dec;25(4):456-‐62.)
Figure
11.Types
of
reactions
used
in
the
MethyLight
assay.
A)
Schematic
representation
of
a
Methylation
dependent
reaction
with
primers
that
cover
CpG
dinucleotides.
B)
Schematic
representation
of
a
Methylation
independent
reaction
with
avoiding
covering
the
CpG
dinucleotides.
[50][51](
(Image:
Trinh
BN1,
Long
TI,
Laird
PW:
DNA
methylation
analysis
by
MethyLight
technology.
Methods.
2001
Dec;25(4):456-‐62.)
27
Chapter
Two
Material
and
Methods
Our
experiments
can
were
carried
out
in
5
major
steps,
which
are
presented
below:
v 2.1:
Tumor
induction
in
mice,
mouse
dissection,
tissue
collection
v 2.2:
DNA
Extraction
from
tissue
samples
v 2.3:
In
vitro
methylation
of
normal
mouse
DNA
v 2.4:
Bisulfite
Conversion
and
recovery
v 2.5:
MethyLight
PCR
(TaqMan
PCR
Reaction
setup)
2.1.
Tumor
induction
in
mice,
mouse
dissection,
tissue
collection:
2.1-‐A.
Tumor
induction
in
the
SCLC
mouse
model
We
have
previously
used
a
SCLC
mouse
model
(received
from
Dr.
Anton
Berns,
Netherland
Cancer
Institute)
in
which
the
Cre/lox-‐based
somatic
conditional
deletion
of
both
alleles
for
Rb1
[55]
and
TP53
[51]
is
achieved
by
intratracheal
Adeno-‐Cre
instillation
[53],
resulting
in
the
induction
of
murine
SCLC.
(Intratracheal
injection
of
Adeno-‐Cre
vector
in
the
mice
was
carried
out
in
our
lab
by
Mario
Pulido)
28
Procedure:
1.
FVB/N
mice
homozygously
floxed
for
Tp53
and
RB,
between
the
ages
of
6-‐12
weeks
are
used.
These
mice
are
old
enough
to
recover
from
the
anesthesia,
and
to
be
intubated
in
the
trachea
with
the
catheter
[53].
Before
starting
the
procedure,
the
mice
are
weighed
to
determine
the
proper
dose
of
anesthesia.
2-‐
Mice
were
anesthetized
by
intraperitoneal
injection
with
a
mix
of
Xylazine
and
Ketamine
as
follows:
we
mixed
150
μL
Xylazine
(100
mg/mL)
with
1mL
Ketamine
(100
mg/mL)
in
8.85mL
of
0.9%
sterile
phosphate
buffered
saline
(PBS)
to
obtain
a
final
concentration
of
100
mg/Kg
Ketamine
and
10
mg/Kg
Xylazine.
We
used
0.075-‐ml/10
g
of
mouse
weight.
We
performed
a
toe
pinch
to
confirm
that
each
animal
was
fully
anaesthetized.
3-‐
We
set
the
mouse
on
a
specially
designed
sloped
support
located
under
a
magnifying
glass
and
close
to
a
light
source.
In
order
to
assure
that
the
mouth
stays
open
during
the
procedure
we
tied
a
thin
thread
to
the
front
teeth
that
was
anchored
firmly
at
the
other
end.
This
way
the
mouth
was
always
open
and
we
could
reach
the
trachea
easily.
We
pulled
the
mouse
tongue
out
in
order
to
open
its
trachea.
This
procedure
was
accomplished
under
a
class
II-‐B2
biosafety
cabinet
in
a
designated
procedure
room.
4-‐
Virus
infection
procedure:
mice
were
infected
with
15
μl
containing
2x10
10
viral
particles
per
mouse
based
on
the
age
of
the
mouse.
[53]
The
Ad-‐Cre
was
prepared
fresh
each
time
and
used
within
one
hour
of
preparation.
For
extended
storage
times,
Adeno-‐Cre
was
stored
at
−
80
°C
or
29
alternatively,
at
4
°C
for
periods
of
a
few
days.
Viruses
were
kept
on
ice
prior
to
infection.
[53]
We
positioned
the
needle
of
the
syringe
into
a
catheter
(dimensions),
and
then
cut
(about
3.5
-‐
4
cm
depends
to
the
length
of
the
syringe
needle)
the
catheter
with
a
blade.
We
then
filled
the
syringe
with
100ul
of
air,
then
we
aspirated
the
virus
solution
into
the
catheter,
and
then
we
added
another
50ul
of
air.
We
wet
the
catheter
is
with
PBS
in
order
to
ease
its
passage
through
the
trachea.
We
next
inserted
the
catheter
in
the
trachea
through
the
open
mouth.
Once
the
catheter
was
inside
the
trachea
we
injected
the
virus
(Figure
11).
We
gently
removed
the
catheter
then
untied
the
thread
and
returned
the
mouse
to
its
cage
to
recover.
The
mouse
began
to
recover
from
anesthesia
after
several
minutes.
Figure
12.
Tracheal
intubation
and
injection
of
Adeno-‐Cre
virus
in
the
mouse.
30
2.1-‐B.
Mouse
tissue
collection
AdenoCre-‐induced
mice
were
euthanized
after
they
showed
serious
symptoms
of
disease
as
determined
by
piloerection,
hunched
posture,
and
reduced
motility
and/or
weight
loss.
By
that
time
most
mice
had
SCLC
in
their
lungs
and
other
parts
of
their
bodies.
This
process
was
done
as
follows:
We
placed
the
mouse
in
a
chamber
linked
to
a
CO2
tank.
We
gradually
increased
the
CO2
flow
until
the
mouse
was
dead.
The
mouse
was
left
in
the
chamber
until
clinical
death
was
determined
(60
seconds
without
seeing
the
mouse
breathing,
and
feeling
the
mouse’s
chest
in
order
to
determine
that
there
was
no
longer
a
heartbeat).
This
was
followed
by
cervical
dislocation
to
ensure
death.
[54].
We
received
remnant
euthanized
FVB/N
and
C57/Bl
strains
for
the
control
DNA
isolation.
We
dissected
the
euthanized
mice
in
order
to
collect
the
needed
organs.
We
collected
liver
and
lung
tissues
from
uninduced
mice,
which
we
used
as
controls.
Freezing
directly
at
-‐80°C
preserved
the
collected
tissues
or
they
were
placed
into
a
plastic
cryomold,
which
was
filled
with
tissue
freezing
compound
(OCT).
We
also
treated
some
of
our
tissue
specimens
with
formaldehyde.
This
method
was
used
to
ensure
the
best
possible
preservation
of
our
tissue
samples.
The
fixed
tissue
were
next
embedded
in
paraffin.
Carcasses
were
disposed
of
by
placing
them
in
a
paper
bag.
We
wrote
date
and
mouse
ID
on
the
bag
and
placed
the
bagged
mice
in
freezer
until
disposal.
31
2.2.
DNA
Extraction
from
tissue
samples
2.2-‐A.
DNA
extraction
from
the
tissues
preserved
in
OCT
or
those
directly
frozen.
2.2-‐A-‐I.
Tissue
Lysis
We
first
prepared
a
lysis
buffer
solution
containing:
Tris-‐HCl
pH
8-‐8.5(100mM),
EDTA
(5mM),
SDS
(0.2%),
NaCl
(200mM),
Proteinase
K
100ug/ml.
The
components
of
the
lysis
buffer
will
break
the
cell
membrane
to
release
the
cytoplasmic
contents
and
will
help
in
the
degradation
of
the
contaminating
proteins.
After
this,
the
SCLC
tissue
pieces
were
removed
from
the
OCT
or
were
thawed
at
room
temperature
(RT).
Small
amounts
(2-‐3
mm
3
)
were
and
placed
in
1.5
ml
micro-‐centrifuge
tubes.
We
added
0.5
ml
of
the
lysis
buffer,
mixed
the
tubes
gently,
and
incubated
the
tubes
at
55°C
overnight.
Next
day,
we
added
more
Proteinase
K
(the
same
amount
as
in
the
previous
day)
thus
doubling
the
final
concentration
of
Proteinase
K.
We
left
the
tubes
at
55°C
for
3-‐4
more
hours.
32
Figure
13.
Two
SCLC
tumor
tissue
specimens
from
FVB/N
induced
mouse
in
melted
OCT.
(Mouse#505)
2.2-‐A-‐II.
Phenol-‐
Chloroform
extraction
We
next
performed
a
two-‐step
purification
using
first
Phenol:
Chloroform,
followed
by
Chloroform.
Phenol-‐
Chloroform
extraction
is
used
to
purify
the
nucleic
acids
and
to
remove
the
proteins
contaminants
from
the
extract.
In
this
technique
the
organic
solvent
solutions
are
mixed
with
equal
volumes
(1:1)
of
the
DNA
solution.
The
proteins
will
partition
into
the
lower
organic
phase
while
the
nucleic
acids
(as
well
as
other
water-‐soluble
compounds
such
as
salts,
sugars,
etc.)
remain
in
the
upper
aqueous
phase
(Figure
14)
Figure
14.
Phase
separation
in
Phenol-‐chloroform
extraction[58].
(Chomczynski,
P.
&
Sacchi,
N:
Single-‐step
method
of
RNA
isolation
by
acid
guanidinium
thiocyanate-‐phenol-‐chloroform
extraction.
Anal.
Biochem.
(1987).
162:
156–159.
doi:10.1016/0003-‐2697(87)90021-‐2.
PMID
2440339)
33
We
return
the
tubes
to
room
temperature
and
added
an
equal
volume
of
the
Phenol/Chloroform/Isoamyl
alcohol
(25:24:1),
solution
to
each
tube,
vortexing
the
samples
for
30
seconds
in
order
to
make
sure
that
the
samples
were
mixed
completely
with
the
phenol:chloroform
solution.
We
spun
the
tubes
for
10
min
at
15
000xg
on
a
tabletop
microcentrifuge.
We
next
removed
the
upper
phase
making
sure
not
to
touch
the
interphase
and
transferred
it
into
a
new
empty
tube.
We
next
added
an
equal
volume
of
Choloroform:Isoamyl
Alcohol
(24:1)
into
each
tube,
vortexed
the
samples
for
30
seconds
and
repeated
the
centrifugation
process
as
described
above.
After
this
we
remove
the
aqueous
upper
phase
and
transferred
it
to
a
new
empty
tube.
Step
3:
DNA
precipitation:
In
this
step,
we
added
1/10
volume
3M
sodium
acetate
in
each
tube,
mixed
and
then
added
2.5-‐3X
volumes
of
100%
ethanol.
Tubes
were
stored
at
-‐20°C
overnight
for
DNA
precipitation.
The
next
day
we
centrifuged
the
DNA
at
16,000xg
for
5
minutes
and
consequently
we
collected
a
DNA
palette
that
collected
at
the
bottom
of
each
tube.
(Figure
15)
Figure
15.
Collecting
DNA
pellet
from
phenol/Chloroform
DNA
extract
with
centrifuge
[58].
(Chomczynski,
P.
&
Sacchi,
N:
Single-‐step
method
of
RNA
isolation
by
acid
guanidinium
thiocyanate-‐phenol-‐chloroform
extraction.
Anal.
Biochem.
(1987).
162:
156–159.
doi:10.1016/0003-‐2697(87)90021-‐2.
PMID
2440339)
34
We
rinsed
the
pellets
with
300µl
70%
Ethanol
to
eliminate
contaminating
salts.
We
centrifuged
the
tubes
again
as
described
above
(5min-‐16000xg),
carefully
removed
the
70%
ethanol.
We
left
the
tubes
open
in
a
chemical
hood
for
about
10
minutes
to
air-‐dry
the
pellets.
We
dissolved
the
DNA
into
100ul
TE-‐4
(Tris
100Mm,
EDTA
5mM).
We
determined
the
DNA
concentration
using
a
Nano
drop
spectrophotometer.
2.2-‐B.
DNA
extraction
from
paraffin-‐embedded
tissues
All
tissue
samples
that
were
paraffin
embedded
were
sent
to
USC
Pathology
Core
for
sectioning
and
slide
preparation.
They
provided
us
with
10
slides
for
each
10µ
thickness,
Hematoxylin
and
Eosin
(H&E)
stained
and
a
coverslipped
H&E-‐stained
slide
for
pathology
review.
Using
the
marked
coverslipped
slide
as
a
giide,
we
located
tumor
tissue
under
the
microscope
and
manually
microdissected
the
sections
to
obtain
cancerous
cells
for
DNA
extraction.
This
was
done
by
another
student
in
our
lab,
Evelyn
Tran.
Figure
16.
Tissues
(samples
from
SCLC
mouse
or
human
patients)
and
paraffin
were
attached
to
the
cassette
formed
a
block,
which
is
ready
for
sectioning.
35
2.3.
In
vitro
methylation
of
normal
mouse
DNA
M.SssI
is
a
bacterial
CpG
methylase
that
methylates
all
CpG
dinucleotides
using
S-‐adenosyl
methionine
(SAM)
as
a
methyl
donor.
M.SssI-‐treated
DNA
is
used
as
a
universally
methylated
reference
sample
in
the
MethyLight
assay.
The
M.SssI
treatment
was
performed
overnight
at
37
o
C
in
a
solution
containing
0.05μg/μL
genomic
DNA,
0.16mM
of
SAM,
1
x
reaction
buffer
(50mM
NaCl,
10mM
Tris-‐HCl,
10mM
MgCl
2,
1mM
DTT,
pH
7.9
at
25°C),
and
0.05
units/μL
of
M.SssI
enzyme.
The
next
day
we
added
a
boost
of
M.SssI
enzyme
and
S-‐adenosyl
methionine
or
SAM
(1/3
of
the
original
volume)
used
for
both
components
together
with
more
water
in
a
total
volume
representing
1/50
of
the
initial
treatment
volume.
We
stored
the
M.SssI-‐treated
DNA
at
+4°C.
We
performed
multiple
treatments.
In
between
treatments,
the
DNA
was
purified
by
Phenol-‐
Chloroform
extraction
followed
by
DNA
precipitation
as
described
above.
2.4.
Bisulfite
Conversion
We
performed
bisulfite
conversion
of
the
genomic
DNA
as
a
first
step
of
the
DNA
methylation
analysis.
In
order
to
accomplish
this
step
we
used
a
commercially
available
kit,
the
EZ
DNA
Methylation
Kit
(ZYMO
Research,
Orange,
CA)
and
followed
the
manufacture’s
instructions
as
follows:
1.
We
first
prepared
the
CT
conversion
reagent
by
adding
750μL
of
water
and
210μL
of
M-‐
Dilution
Buffer
to
one
tube
containing
the
CT
conversion
reagent
and
vortexing
every
1–2min
36
for
a
total
of
10
min.
2.
We
next
added
5μL
of
the
M-‐Dilution
Buffer
to
each
of
the
DNA
samples
and
adjusted
the
total
volume
to
50μL
with
sterile
water.
We
mixed
the
sample
by
flicking
the
tubes
or
by
pipetting
up
and
down.
We
then
incubated
the
sample
at
37°C
for
15
min.
[48]
3.
After
this
incubation,
we
added
100μL
of
the
prepared
CT
Conversion
Reagent
to
each
sample
and
vortexed
for
complete
mixing.
4.
We
incubated
the
samples
in
a
thermo
cycler
machine
using
the
following
conditions:
95
°C
for
30
s,
50
°C
for
60min,
repeated
for
16
cycles.
At
the
end
we
incubated
the
sample
on
ice
for
10min.
5.
Next
day
we
cleaned
up
the
DNA
by
passing
it
through
a
column
provided
by
the
manufacturer.
We
first
added
400μL
of
M-‐Binding
buffer
to
the
sample
and
mixed
by
pipetting
up
and
down.
Then
we
loaded
the
sample
onto
a
Zymo-‐Spin
I
column
and
placed
the
column
into
a
2-‐mL
collection
tube.
[48].
The
loaded
columns
were
centrifuged
at
full
speed
(>10,000g)
or
(15000xg)
for
30
s.
The
flow
through
was
discarded.
6.
We
added
200μL
of
M-‐Wash
Buffer
to
the
column
and
centrifuged
the
columns
at
full
speed
(15000xg
for
30
s).
7.
We
added
next
200μL
of
M-‐Desulphonation
Buffer
to
the
column
and
kept
the
column
at
37
room
temperature
for
15
min,
then
spun
it
down
at
15000xg
for
30
s.
8.
We
did
another
wash
by
adding
200μL
of
M-‐Wash
Buffer
to
the
columns
and
then
centrifugation
at
15000xg
for
30
s
We
did
another
final
wash
by
adding
200μL
of
M-‐Wash
Buffer
and
centrifugation
at15000xg
for
2
min.
9.
We
eluted
the
samples
by
adding
10μL
of
M-‐Elution
Buffer
directly
to
the
column
matrix,
[48]
then
spun
the
samples
into
1.5ml
microcentrifuge
tubes
at
15k
rcf
,
for
1
min.
The
bisulfite-‐
converted
DNA
was
stored
at
−20
o
C
until
it
was
used
for
MethyLight
reactions.
[48]
(Figure
13)
Figure
17.
Conversion
of
non-‐methylated
cytosine
by
DNA
Bisulfite
conversion
(Figure
by:
ZYMO
research)
38
2.5.
MethyLight
Analysis
The
MethyLight
assay
makes
use
of
the
TaqMan
PCR
principle,
which
requires
forward
and
reverse
primers
as
well
as
a
non-‐extendable
oligomeric
probe,
which
emits
fluorescence
only
after
it
is
degraded
by
the
5→3
exonuclease
activity
of
the
Taq
polymerase.
[48]
Figure
18.
Schematic
procedure
of
the
Quantitative
PCR
(Image:
EpiQuik™)
For
each
PCR
reaction,
we
used
the
same
basic
reaction
setup
with
different
primers/probe
sets.
Each
individual
PCR
reaction
contained
10μL
DNA,
15.4μL
PreMix
Solution,
4.5μL
OligoMix
Solution
(1.5μL
of
each
primer
and
probe),
and
0.1μL
Taq
polymerase
in
a
total
of
30μL
PCR
volume.
[48]
The
premix
solution
contains:
MgCl2
(3.5
mM),
1
x
Taq-‐
Man
Buffer,
1
x
TaqMan
39
stabilizer
(enables
to
maintain
the
activity
of
pre
mixture
for
over
a
month
even
when
stored
at
room
temperature,
or
over
two
years
in
the
freezer)
and
0.2mM
of
each
dNTP.
The
OligoMix
is
the
mixture
of
the
two
primers
and
probes
that
have
been
diluted
such
that
every
reaction
4.5ul
contains
(1.5ul
of
each
of
the
primer
and
the
probe
at
a
concentration
of
2µM
for
each
primer
and
0.67µM
for
the
probe.
The
combined
PreMix
Solution,
OligoMix
Solution,
and
Taq
Gold
polymerase
for
each
reaction
is
referred
to
as
the
MasterMix
Solution.
[48]
We
loaded
10μL
of
bisulfite
converted
DNA
plus
20μL
of
the
MasterMix
Solution
in
each
PCR
well.
Individual
OligoMix
Solutions
were
prepared
for
the
gene
investigated
by
MethyLight
or
any
other
quality
control
reactions
used
in
the
analysis.
MethyLight
assays
used
were
previously
developed
in
the
Laird
and
Laird-‐
Offringa
labs
based
on
data
obtained
from
other
studies.
These
assays
were
specific
for
mice
loci
overlapping
the
same
genomic
regions
as
in
humans.
Each
MethyLight-‐based
data
point
is
the
result
of
the
combined
analysis
of
a
methylation-‐
dependent
PCR
reaction
and
methylation-‐independent
PCR
reaction
on
reference
(M.SssI-‐
treated
DNA)
and
experimental
DNA
samples.
[48]
The
results
are
expressed
as
PMR.
The
figure
bellow
shows
how
to
calculate
PMR.
Figure
19.
PMR
(Percent
of
Methylated
Reference)
calculation.
40
All
PCR
reactions
were
carried
out
as
follows:
one
cycle
at
95
o
C
for
10min
followed
by
50
cycles
at
95
o
C
for
15
s,
and
60
o
C
for
1
min.
[48]
The
MethyLight
results
are
expressed
in
the
following
chapter.
41
Chapter
Three
Results
3-‐A.
SCLC
induction
in
FVB/N
mice
and
tissue
specimen
collection
from
induced
and
un-‐induced
FVB/N
mice
and
C57
mice
We
collected
seven
tumors
from
5
FVB/N
induced
mice
and
normal
lung
tissue
from
an
un-‐
induced
FVB/N
mouse.
We
also
obtained
two
liver
tissues
(one
male
one
female)
from
un-‐
induced
FVB/N
mice
and
two
liver
tissues
(one
male
one
female)
from
remnant
euthanized
C57Bl/6
mice.
Tumors
were
stored
in
OCT.
Tumors
(24)
from
FVB/N
mice
that
were
fixed
in
formalin
and
then
embedded
in
paraffin
were
also
analyzed.
The
normal
tissues
were
all
fresh
frozen
and
stored
at
-‐80°C.
(Table
1)
42
Table
1.
Summary
of
the
tissue
samples
used
in
the
MethyLight
analysis.
Mouse&ID
Adeno,Cre&
Induction Mouse&type Sample&type
Microdisection&
status
3525 No C57Bl/6 Normal/Liver/Tissue No
3527 No C57Bl/6 Normal/Liver/Tissue No
NA No FVB/N Normal/Liver/Tissue No
NA No FVB/N Normal/Liver/Tissue No
506 yes FVB/N SCLC//Lung/Tumor No
505 yes FVB/N SCLC//Lung/Tumor No
505 yes FVB/N SCLC//Lung/Tumor No
499 yes FVB/N SCLC//Lung/Tumor No
486 yes FVB/N SCLC//Lung/Tumor No
483 yes FVB/N SCLC//Lung/Tumor No
483 yes FVB/N SCLC//Lung/Tumor No
887 No FVB/N Normal/Lung/Tissue No
299B300NI No FVB/N Normal/Lung/Tissue yes
302NI No FVB/N Normal/Lung/Tissue yes
304LNI No FVB/N Normal/Lung/Tissue yes
247LNI No FVB/N Normal/Lung/Tissue yes
148N yes FVB/N Normal/Lung/Tissue yes
165N yes FVB/N Normal/Lung/Tissue yes
176LN yes FVB/N Normal/Lung/Tissue yes
214AN yes FVB/N Normal/Lung/Tissue yes
219N yes FVB/N Normal/Lung/Tissue yes
348LN yes FVB/N Normal/Lung/Tissue yes
369LN yes FVB/N Normal/Lung/Tissue yes
281BN yes FVB/N Normal/Lung/Tissue yes
150LN yes FVB/N Normal/Lung/Tissue yes
214BN yes FVB/N Normal/Lung/Tissue yes
369LT yes FVB/N SCLC//Lung/Tumor yes
281BT yes FVB/N SCLC//Lung/Tumor yes
148T yes FVB/N SCLC//Lung/Tumor yes
150LT yes FVB/N SCLC//Lung/Tumor yes
165T yes FVB/N SCLC//Lung/Tumor yes
176LT yes FVB/N SCLC//Lung/Tumor yes
214AT yes FVB/N SCLC//Lung/Tumor yes
214BT yes FVB/N SCLC//Lung/Tumor yes
219T yes FVB/N SCLC//Lung/Tumor yes
348LT yes FVB/N SCLC//Lung/Tumor yes
43
3-‐B.
In
vitro
DNA
methylation
(M.SssI
treatment)
Results:
We
used
DNA
extracted
from
normal
liver
tissues
from
FVB/M
un-‐induced
mice
and
C57Bl/6
mice
to
perform
in
vitro
DNA
methylation
using
the
M.SssI
methyltransferase.
(Table
1)
Since
we
intended
to
treat
a
large
amount
of
DNA
we
decided
to
perform
this
treatment
multiple
times
using
lower
amounts
of
enzyme
(see
Materials
and
Methods).
After
each
treatment,
we
set
aside
small
aliquots
for
future
testing.
Between
treatments
we
cleaned
up
the
DNA
by
standard
phenol/chloroform
extraction
followed
by
DNA
precipitation.
After
3
(FVB/N
mice)
and
4
(C57/Bl6)
consecutive
M.SssI
treatments,
we
compared
the
DNA
methylation
levels
in
samples
collected
after
each
treatment
with
a
sample
that
was
previously
treated
using
large
amounts
of
M.SssI
enzyme
and
which
was
assumed
to
be
a
fully
methylated
sample
(this
sample
was
provided
by
Dr.
Daniel
Weisenberger).
We
bisulfite
converted
about
0.5ug
of
DNA
collected
after
each
treatment
and
performed
MethyLight
assays
using
primers
for
a
control
reaction
(methylation
independent
reaction
that
is
targeted
to
a
mouse
repeat
regions
does
not
cover
any
CpGs
,MB-‐111
(gene
ID)
,
and
reactions
for
three
gene
IDs
(MB-‐113,
MB-‐115,
MB-‐123)
single
copy
gene
promoters
(methylation
dependent
reactions
since
these
reactions
cover
CpGs).
44
Figure
20.
Evaluation
DNA
methylation
levels
by
ΔCt
determination.
(Reaction
Image:
Image:
Trinh
BN1,
Long
TI,
Laird
PW:
DNA
methylation
analysis
by
MethyLight
technology.
Methods.
2001
Dec;25(4):456-‐62.)
For
the
evaluation
of
the
methylation
levelS
of
sample
DNA,
unlike
the
classic
MethyLight,
we
only
determined
the
Cts
(cycle
threshold)
for
each
of
these
reactions.
We
compared
the
difference
(ΔCt)
between
the
Ct
for
the
single
copy
gene
and
the
Ct
of
the
control
reaction.
There
was
a
decrease
in
the
delta
Ct
with
each
treatment
suggesting
that
the
DNA
became
more
methylated
with
each
treatment.
However,
after
three
treatments
the
ΔCts
for
FVB/N
were
still
higher
than
those
calculated
for
the
positive
control
sample
we
assumed
to
be
fully
methylated
(Table
2).
We
decided
to
do
another
round
of
treatment
for
these
DNAs
using
a
larger
amount
of
M.SssI
enzyme.
The
delta
Ct
of
the
sample
treated
four
times
(FVB/N)
was
lower
than
that
of
the
sample
that
was
assumed
to
be
fully
methylated
(positive
control)
for
45
the
MB-‐115
and
MB-‐123
reactions
(Table
3).
These
last
treated
samples
were
used
further
in
the
MethyLight
analysis
as
methylated
references.
We
treated
the
C57/Bl6-‐derived
DNA
four
times
with
M.SssI
enzyme
before
checking
the
ΔCts.
For
comparison
we
used
the
last
treated
sample
of
the
male
FVB/N
mouse.
Unfortunately,
the
DNA
methylation
levels
for
the
same
reactions
(MB-‐115
and
MB-‐123)
were
still
low
compared
with
sample
and
they
did
not
change
even
after
an
additional
treatment
with
higher
levels
of
M.SssI
enzyme.
(Table3)
Table
2.
FVB/N-‐
M-‐SssI
first
Treatment
results.
First
group
of
our
treatments
delta
C
(t)
results
(Positive
control:
assumed
Fully
Methylated
Sample)
Table
3.
FVB/N-‐
M-‐SssI
second
Treatment
results.
Adding
the
fourth
treatment
(Tr-‐4)
to
our
previous
group
with
more
enzyme
(FVB/N)
Probe Tr-1 Tr-2 Tr-3 Positive Control
MB-113
Male 12.3 11.5 11
Female 14 12 12
Positive Control 10
Tr-1 Tr-2 Tr-3 Tr-4
Positive
Control
MB-115
Male 12 12 12 11
Female 14 13 12 11
Positive
Control 11
MB-123
Male 12 11 11 10
Female 13.5 12 11 10
Positive
Control 10.5
46
Table
4.
C57Bl/6-‐
M-‐SssI
Treatment,
First
determination
(Calculated
delta
C(t)s
between
MB-‐111
gene
ID
the
group
of
genes
IDs
of
interest
MB-‐113
and
MB-‐123)
Table
5.
C57BL/6-‐
M-‐SssI
Treatment
results,
Second
determination.
Probe Tr-1 Tr-2 Tr-3 Tr-4 Tr-5 FVB/N
MB-113
Male 13.5 12 11 12 13 11
Female 14 12 12 11 12.5 11
MB-123
Male 14.5 11 10 9.5 9.5 9.5
Female 15 12 10 9 10 9
47
3-‐C.
MethyLight
analysis
Results
for
microdissected
SCLC
tumors:
After
obtaining
the
delta
C(t)s
from
our
fresh
and
frozen
SCLC
mouse
samples
we
did
our
experiment
on
SCLC
paraffin
embedded
tissues,
from
which
tumor
tissue
had
been
obtained
by
micro-‐dissection.
Table
6:
PMR
values
from
paraffin
embedded
tissues
(Micodissected)
Mouse&ID
Adeno,Cre&
Induction Sample&type
MB,111&
C(t)
MB,124&
PMR
MB,123&
PMR
MB,41&
PMR
MB,114&
PMR
MB,115&
PMR
MB,121&
PMR
MB,125&
PMR
MB,117&
PMR
299#300NI no Normal.Lung.Tissue 17.2 0 0 8 0 3 6 0 105
302NI no Normal.Lung.Tissue 17.1 0 0 5 1 1 6 1 113
304LNI no Normal.Lung.Tissue 17.2 0 0 9 2 6 7 NA 111
247LNI no Normal.Lung.Tissue 17.1 0 0 8 3 11 16 0 173
148N yes Normal.Lung.Tissue 16.2 0 0 5 0 3 9 NA 105
165N yes Normal.Lung.Tissue 16.7 0 0 1 0 7 6 0 66.4
176LN yes Normal.Lung.Tissue 17.6 0 0 4 0 5 15 NA 111
214AN yes Normal.Lung.Tissue 18.7 0 0 17 0 3 13 0 183
219N yes Normal.Lung.Tissue 18.1 0 0 9 0 0 23 NA 186
348LN yes Normal.Lung.Tissue 18.1 0 0 4 0 1 8 0 162
369LN yes Normal.Lung.Tissue 17.1 0 0 8 4 2 10 NA 171
281BN yes Normal.Lung.Tissue 17.1 0 0 3 4 0 10 NA 183
150LN yes Normal.Lung.Tissue 17.1 0 0 5 0 4 10 NA 104
214BN yes Normal.Lung.Tissue 18.1 0 0 6 0 12 28 NA 133
369LT yes SCLC..Lung.Tumor 17.2 0 0 13 0 4 12 NA 108
281BT yes SCLC..Lung.Tumor 18.1 0 0 14 0 37 12 NA 188
148T yes SCLC..Lung.Tumor 15.7 6 5 2 31 13 193 NA 67.1
150LT yes SCLC..Lung.Tumor 17.1 4 7 13 0 15 16 NA 148
165T yes SCLC..Lung.Tumor 16.1 12 8 28 13 21 4 NA 181
176LT yes SCLC..Lung.Tumor 16.6 0 0 11 0 10 19 0 114
214AT yes SCLC..Lung.Tumor 16.1 5 3 39 0 4 6 NA 106
214BT yes SCLC..Lung.Tumor 15.2 6 5 10 14 16 5 0 107
219T yes SCLC..Lung.Tumor 17.2 0 0 12 2 10 14 NA 188
348LT yes SCLC..Lung.Tumor 15.2 6 2 3 1 5 2 NA 108
48
3-‐D.
MethyLight
analysis
Results
for
SCLC
tumors
that
were
not
microdisected:
We
performed
ML
analysis
on
seven
tumors
DNA
and
one
normal
lung
tissue
DNA
that
were
not
microdissected.
The
results
are
expressed
as
PMRs
and
shown
in
Table
7.
Table
7.
PMR
valuess
from
MethyLight
reaction
on
7
SCLC
tumors
DNA
and
one
normal
lung
DNA.
Mouse&ID
Adeno,Cre&
Induction Sample&type
MB,111&
C(t)
MB,114&
PMR
MB,53&
PMR
MB,125&
PMR
MB,124&
PMR
MB,115&
PMR
MB,123&
PMR
MB,121&
PMR
MB,41&
PMR
MB,117&
PMR
506 yes SCLC**Lung*Tumor 18.3 0 0 0 0 0 0 1 1 115
505 yes SCLC**Lung*Tumor 16.3 0 0 0 0 0 0 0 0 108
505 yes SCLC**Lung*Tumor 15.0 0 0 0 0 0 1 1 1 70
499 yes SCLC**Lung*Tumor 15.2 0 0 0 0 0 0 1 0 42
486 yes SCLC**Lung*Tumor 15.1 0 0 0 0 1 0 35 1 26
483 yes SCLC**Lung*Tumor 16.3 0 0 0 0 0 0 0 1 14
483 yes SCLC**Lung*Tumor 15.1 0 0 0 0 0 0 1 0 31
887 no Normal*Lung*Tissue 16.1 0 0 0 0 0 0 1 1 43
49
Table
8.
DNA
methylation
differences
in
frozen
tissues
and
fresh
frozen
paraffin
embedded.
In
this
figure
we
compared
the
MethyLighht
PMR
values
between
different
gene
IDs
from
tissues
that
were
either
microdissected
from
paraffin
blocks
or
not
microdissected
from
frozen.
50
Chapter
Four
Discussion
We
took
advantage
of
the
SCLC
mouse
model
to
test
whether
DNA
methylation
markers
that
were
detected
in
the
blood
of
human
patients
with
SCLC
are
also
methylated
in
the
SCLC
tumors
that
developed
in
these
mice.
For
this,
I
assisted
Mario
Pulido
in
inducing
SCLC
tumors
in
mice
with
floxed
Tp53
and
Rb
alleles.
I
extracted
DNA
from
fresh
frozen
normal
and
tumor
lung
tissues
obtained
from
these
mice.
DNA
was
also
extracted
from
normal
and
tumor
tissues
microdissected
from
paraffin-‐embedded
tissue
sections
by
Evelyn
Tran.
These
DNAs
were
analyzed
by
MethyLight
using
mouse
probes
for
methylated
DNA
regions
detected
in
the
blood
of
SCLC
patients.
Since
MethyLight
analysis
requires
a
fully
methylated
reference
sample,
I
generated
large
amounts
(3-‐5μg)
of
such
reference
DNA
by
in
vitro
methylating
normal
mouse
DNA
from
the
two
mouse
strains
most
commonly
used
for
lung
cancer
studies.
It
is
important
to
have
control
DNA
matched
to
each
strain
to
avoid
biases
due
to
genetic
differences
between
these
inbred
strains,
which
might
affect
probe
and
primer
hybridization.
Having
a
fully
methylated
reference
DNA
(>10ug)
is
very
important
when
doing
a
MethyLight
assay
since
an
incomplete
methylated
reference
DNA
could
result
in
erroneous
PMRs.
It
is
preferable
to
produce
large
amounts
of
this
reference
sample
since
generating
such
reference
DNA
is
a
laborious
procedure
and
because
the
level
of
DNA
methylation
could
vary
from
one
51
treatment
to
another.
This
way,
the
reference
can
be
used
for
many
MethyLight
assays
and
variability
in
the
PMR
results
due
to
batch
differences
can
be
avoided.
Treatment
of
large
amounts
of
DNA
required
the
use
of
large
amounts
of
enzyme
as
suggested
by
the
manufacturer
of
the
enzyme.
In
order
to
reduce
the
cost
of
the
treatment
we
decided
to
treat
the
DNA
multiple
times
with
reduced
amounts
of
enzyme.
The
effects
of
the
treatments
were
evaluated
by
calculating
delta
Cts
between
methylation-‐dependent
reactions
and
methylation
independent
reactions.
Because
three
successive
treatments
did
not
bring
the
methylation
levels
to
the
levels
of
a
positive
control
we
performed
another
treatment
with
higher
levels
of
enzymes.
For
one
of
the
C57/Bl6
strains
we
needed
to
perform
the
5
th
treatment.
For
this
strain
the
levels
of
methylation
did
not
decreased
even
after
an
extra
treatment
with
higher
levels
of
enzyme
and
the
Cts
remained
lower
that
the
positive
control.
This
could
be
due
to
differences
in
DNA
sequences
between
the
two
strains
(polymorphisms)
that
can
result
in
differences
in
the
efficiency
of
the
MethyLigt
assays.
During
the
course
of
the
successive
treatments
with
M.SssI
enzyme
we
determined
that
there
was
no
need
to
purify
the
DNA
between
each
treatments
but
simply
add
more
enzyme
and
SAM
substrate
(data
not
shown).
This
way
we
prevented
DNA
from
being
lost
during
the
clean
up
steps.
The
results
from
testing
DNA
methylation
markers
in
DNA
isolated
from
microdissected
tissue
and
from
fresh
frozen
tissues
not
subjected
to
microdissection
showed
differences
in
DNA
methylation
levels
for
these
markers.
This
could
be
due
to
the
reduced
amount
of
tumor
tissue
in
the
fresh
frozen
specimens
and
contamination
with
normal
tissue.
Data
from
microdissected
tissue
will
of
course
be
more
reliable.
Alternatively,
it
is
possible
that
the
frozen
tissue
was
not
52
well
preserved
during
DNA
isolation,
possibly
causing
DNA
degradation.
These
tissues
will
need
to
be
further
studied.
We
did
not
detect
an
increase
in
the
DNA
methylation
levels
in
the
mouse
tumors
for
all
the
MethyLight
assays
we
have
tested,
nor
for
all
the
tumor
samples.
This
can
be
due
to
differences
between
the
mouse
and
human
genomes.
Also,
human
cancers
take
20-‐40
years
to
develop
while
the
tumors
in
the
mouse
model
are
driven
by
specifically
activated
genetic
events
(AdenoCre
induction)
and
might
develop
too
quickly
to
acquire
the
same
methylation
changes.
A
genome-‐wide
DNA
methylation
analysis
of
mouse
SCLC
tumors
would
be
very
useful
to
determine
the
presence
of
DNA
methylation
in
the
tumors,
since
our
current
choices
of
probes
were
based
on
human
lung
cancer
data.
If
regions
are
found
to
be
consistently
methylated
in
tumors,
new
MethyLight
probes
could
be
developed.
One
problem
is
that
the
cell
of
origin
of
SCLC
is
the
pulmonary
neuroendocrine
cell,
which
is
very
difficult
to
purify
and
analyze.
Thus,
it
is
difficult
to
exclude
that
any
DNA
methylation
see
in
tumors
is
new
methylation
not
present
in
the
cell
of
origin.
Nevertheless,
regions
that
are
consistently
methylated
in
tumors
(even
if
methylated
in
cell
of
origin)
would
likely
be
of
use
for
detection,
since
SCLC
tumors
are
necrotic
and
shed
much
DNA
into
the
blood
stream.
53
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Abstract (if available)
Abstract
Lung cancer is the top cancer killer in the United States. This cancer starts in the cells lining the bronchi or other parts of the lung such as bronchioles or alveoli. It can be classified into two major groups: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Although SCLC is less common than NSCLC it is the most aggressive type of lung cancer. To this day, there are no effective serum biomarkers for detection, prediction of response or monitoring this disease. Such markers could be very valuable because currently most SCLC patients are diagnosed after their cancer has metastasized, and there is no way apart from imaging to test for response or recurrence. ❧ It has been amply demonstrated that epigenetic modifications, such as alterations in cytosine-5 DNA methylation, occur frequently in all types of cancer. DNA methylation can be detected in the serum of cancer patients using a sensitive assay called MethyLight. Our lab has developed blood-based DNA methylation markers for NSCLC that have also been shown to be present in the blood of small cell lung cancer patients. We were interested to determine whether these markers are also present in tumors derived from a mouse model of SCLC. These mice are important tools to develop new therapies, and identify molecular markers that could be used to determine whether the mice are responding to treatment or showing recurrent disease would be very useful. ❧ We extracted the DNA from normal and tumor tissue from mice with SCLC and disease-free control mice. Prior to DNA extraction, tissues were either microdissected from paraffin blocks or were directly taken from a frozen specimen. We performed MethyLight analysis on these DNAs and compared the methylation levels between normal lung and tumors. In order to setup the MethyLight assay for mouse DNA, we required a (positive control) fully in vitro methylated DNA sample to serve as reference in the MethyLight assay. We generated large amounts (3-5μg) of in vitro methylated DNA for two different strains of mice. This DNA is an important resource for the lab since it can be used as a positive control MethyLight assays for years to come, and eliminates the biases associated with using different batches of reference DNA. ❧ MethyLight analysis of normal/tumor DNA from the SCLC mouse model showed increased methylation in some of the tumors when the microdissected material was used, but little or no methylation when the frozen samples were examined. These results suggest that microdissection might be important in DNA methylation analysis. Because modest levels of methylation were detected but no marker showed high sensitivity, we suggest that a genome-wide analysis of DNA methylation patterns in these mice would be useful. It might uncover DNA methylation abnormalities that are specific for mice with SCLC.
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University of Southern California Dissertations and Theses
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Asset Metadata
Creator
Alef-Omidy, Honey
(author)
Core Title
DNA methylation markers for blood-based detection of small cell lung cancer in mouse models
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
11/05/2014
Defense Date
10/23/2014
Publisher
University of Southern California
(original),
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Tag
biomarkers,DNA methylation,MethyLight,mouse models,OAI-PMH Harvest,small cell lung cancer,sodium bisulfite
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Laird-Offringa, Ite A. (
committee chair
), Tokes, Zoltan A. (
committee member
), Weisenberger, Daniel (
committee member
)
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alefomid@usc.edu,honey_alefomidi@yahoo.com
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Tags
biomarkers
DNA methylation
MethyLight
mouse models
small cell lung cancer
sodium bisulfite