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Cycling
Characteristics
Human
Lymphocytes
By Lewis M. Schiffer,
Arnold
M. Markoe,
and
The
cycling
characteristics
ripheral
blood
of
hemagglutinin-stimulated
examined
by means
employing
assay
the
nuclei
contain
DNA
exogenous
primer-template
capability.
by
D
ESPITE
THE
mitogenic
measure
ofthe
fraction
Reliance
upon
the
for
during
the
according
quiescent
or noncycling
appear
of
to change
assay
procedure,
and
enous
merase
primer-template
activity
does
malian
cell populations.4
These
studies,
polymerase
addition
of the
during
all utilizing
activity
From
the
Cell
and
for
Submitted
accepted
August
January
Supported
Biology
the
and
Western
Address
©
1974
Blood,
Vol.
23.
a Clinical
Cancer
reprint
General
by Grune
44,
during
of
be
loss
rather
of
than
as
been
no
in vitro
index
incubation.
DNA
cycle”2
and
enzyme
activities,
cycle”2’4
polymerase
little
activity
however,
may
system,
measure
studied
cohort
in this
upon
an
and
endog-
cell nor the proportion
of cells
We have
recently
described5
Laboratories.
Allegheny
Montefiore
Hospital.
November
General
Hospital.
Pittsburgh.
12.
1973;
second
Center
Grant
polymam-
average
cannot
not
detect-
added
is placed
in
do
remain
is not
synthesis
cell-free
primer-template.
exhibits
marked
shows
and
sufficient
using
DNA
enzyme
a
reliable
is not
investigated
primer-template
of the
revision
No.
Radiation
Research
Pennsylvania
for
has
labeling
cell
assay
cells
Biology
first
virtue
nuclear
DNA-dependent
DNA
fluctuation
in synchronized
a cell-free
of Medicine.
1973;
may
DNA
indicate
in a popuan in vitro
Pittsburgh.
Pa.
Pa. 15213.
revision
January
28.
/974:
29. 1974.
by
and
Allegheny
Radiation
G,
that
divisions
phytohemagglutinin
point
DNA
present,
considerable
in all of the
The Department
cell
the
the activity
of the enzyme
in a given
lation
which
exhibit
enzyme
activity.
152/2.
by
there
of exogenous
cytoplasmic
DNA
reliance
being
exhibit
DNA
polymerase.
actively
Nuclear
Ifexogenous
sole
of
time,
hypothesis
first
DNA-dependent
been
in the
appreciably
cells.’
at any
has
systems.”3
in quiescent
onset
cycle
availability,
of
(3HTdR)
cellular
cycle
to position
able
cycle
lymphocytes,
in cycle
assay
systems
which
require
the
It would
appear
that
the activity
fluctuation
the
of the
by loss of DNA
thymidine
cell
to the
of cell
support
from
application
variation
mammalian
S. R. Nelson,
in-
human
ofcells
prior
primer-template
fraction
for
tritiated
this purpose.
In recent
years,
to
cells
DNA
EXTENSIVE
stimulus
presented
retired
techniques
1 hr
Estimates
daughter
utilizing
The
these
Janet
time, and fraction
of cycling cells are made
for the first
4 days in culture.
Evidence
is
whose
endogenous
of
labeled
cells
5-1
synthesis.
methods
of
polymerase
and/or
cells
creases
phyto-
cultures
were
of a new technique
fraction
Winkelstein,
M. Mikulla
pe-
in
radioautographic
which
Alan
John
human
lymphocytes
of
In Vitro
Chapter
requests:
Hospital.
1974
Grant
Research
CA 05224
of The A rthritis
Lewis
320 East
& Stratton.
1 (July),
Therapy
Training
M.
North
Schiffer.
A venue,
.
both
from
Foundation
M.D..
the
CA 10438.
National
and
Cancer
a Radiation
Institute
and
(A. W.).
Cell
Pittsburgh,
and
Radiation
Biology
Laboratories.
Pa. 15212.
Inc.
99
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
SCHIFFER
100
radioautographic
method
dependent
DNA
dogenous
nuclear
primer-template
allows
nuclei
for
the determination
are PDP-positive,
The
fact
merase
that
and
in DNA
are
are
to label
forced
It is appropriate
tamed
from
in
5-180
DNA
ascites
cells
PDP-negative.
(PHA)
become
These
same
PDP-positive
cells,
by S-l80
measured
PDP-negative
PDP-positive,
most
always
not
figures
positive
been
we
feel
period,
such
the proliferative
and
they
are
as murine
pool.
periods,
should
zyme or DNA
are
It has been
PDP-positive
Cultured
they
heat
with
decreasing
but
generally
the
PHA.
By
of
of the
the
assay
to
cell’s
own
in addition
contain
referred
DNA-dependent
to as the DDDP
DNA
index.
estimation
of
lack of
state.
cytokinetic
of
cycle,
the
DNA
and
similar
at
period
state
human
G,
en-
by
murine
some
PHA.9”
peripheral
of the PDP
index
provide
data
as
stimulated
by
primer-template
of cells
can
to
G,
may
be
relationships
of the
labeling
index.
This
parameters
a short
polymerase
a short
fraction
alone
cells
of cells whose
nuclei
cells in cycle
(GF)
in
exogenous
the
G2
if they
are in
or are in long G,
in lymphocytes
that
50%
al-
This finding
reinforces
of the cell cycle.
Determination
therefore,
DNA,
Dead
prophase
with
the quiescent
to suggest
that
interface
are
become
mitosis.7
very
time
of cycle.
synthesis,
polymerase
time
PDP
blood
of
positive
cells
all
is ob-
approximately
with
prophases
of
the fraction
fraction
of
cycle
ensure
the
Peripheral
during
Thus,
that
the
out
evidence
In this paper
we shall
report
the time
indices
in comparison
with
the 3HTdR
mits
with
out
evidence
characteristics
0,-S
In
LI.
by 45#{176}C
temperatures,
the
of
cell
those
information
normally
positivity
have
a short
to the
utilized
culture.
killed
of
poly-
than
into cycle
by phytohemagglutinin
to the start
of the S phase.
from
laboratory
estimate
have
structure
modification
This
have a short
G, phase
duration.8
positivity
during
the various
phases
also
3HTdR
should
all be PDP-positive
out of cycle (G0 period),
have
been
stimulated
there
is considerable
intimate
measuring
PDP-positive.
blood
lymphocytes
are normally
out
in relationship
to the onset
of DNA
to
we
are
DNA
constituents.
the
of 30 mm.7
Mitoses
are
which
cell system
is used,
lymphocytes
they
these
enthe
It
whose
other
exceeds
which
directly,
by this
is a close
human
time
after
Furthermore,
cells
contain
Cells
are
DNA-
population
by hydroxyurea.6
paper,
not label,
either
because
not being in a priming-template
shown
in that
PDP-positive.
tumor
cells,
Cells that are
murine
tumors
which
the estimates
of PDP
tumors
ascites
measured
believe
cells stimulated
5-1 1 hr prior
with
a half-time
depending
upon
have
we
are
of
the cell’s
own
assay
is called
(PDP)
assay.
they
contain
index
synchronized
in this
that
if they
what
synthesis
as described
indication
PDP
presence
in a given
as primer-template.
the
to consider
cells
lymphocytes
to date,
the
utilizing
in vitro
polymerase
of cells
is an
to act
performed
All
labeled
competent
synthesis
index.
detecting
nuclei,
This
DNA
of the fraction
the PDP
index.
nuclei
DNA
experiments
qualitatively
polymerase
in individual
DNA
as primer-template.
available,
DNA-dependent
ET AL
various
whose
estimated.
is
nuclei
This
PDP
and
information
DDDP
per-
intervals
during
is
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
HUMAN
101
LYMPHOCYTES
MATERIALS
Lymphocyte
From
METHODS
Cultures
60 to
individuals
80
with
ml
of blood,
their
a modification
after
with
informed
of the
pensions,
from
each
large
20%
calf
mg
sample.
Each
being
All
per
hypaque
contained
by
3HTdR
in
was
x
100
PHA
were
blood,
were
received
by
for
Multiple
of
from
RPMI
and
100
a freshly
using
isopaque.’2
Cell
were
1640
5%
sus-
prepared
supplemented
sg/ml
streptomycin.
prepared
in a water-saturated
normal
centrifugation
cultures
in S ml
penicillin,
0. 1 ml
incubated
processed
substituted
106 cells
U/mI
was
obtained
lymphocytes.
3.0
1-glutamine,
cultures
ml
cells
85%-90%
culture
zg/ml
stimulated
(Burroughs-Wellcome).
in that
contained
300
heparin
Mononuclear
B#{216}yum technique
serum,
cultures
0.125
consent.
centrifugation,
with
Those
AND
stock
CO2
solution
atmosphere
at
3TC.
Labeling
3HTdR
Cultures
37’C
received
in
air.
of NTB2
(L.I.)
grains
Cultures
dried.
disrupt
made
to a slight
by affixing
medium
Ficoll
The
the
per
remainder
The
procedure
DNase-activated,
tion
of calf
thymus
the
DDDP
Transforming
for
incubated
slides
were
cells.
Background
for
with
developed
and
was
45
a
mm
stained,
and
minimal,
the
and
M
twice,
only
PDP
slide,
each
nuclei
assay
and
of
at
spread
and
ml
The
to
use
medium,
slides
fixation,
were
previously.1
of
M
the
lymphocyte
and
and
nuclei
slide.
cells
The
medium
MgCl2,
then
counted
slides
air
to
were
then
chambers
was
and
lO_2
was
supplemented
M
KCI
activity,
fl-mercaptoethanol
were
added.
This
in 8.3
x
with
400
lO_2
mg
17- 19 Ci/mmole;
was
added
was
identical
LI.
Figure
to a final
for 45 mm.
radioautographic
were
and
appears
deoxyguanosine-5’-tri-
medium
at 37’C
process
Briefly,
incubation
(specific
incubated
washing,
hundred
the
slides,
This
deoxycytidine-5’-triphosphate,
Prior
of incubation
M.
acid-cleaned
dried.
to
described
0.5
to
air
adherent
lO2
25’C).
on
again
procedure
to determine
the
PDP
to
the
1 shows
nuclei.
Index
for
the
DDDP
sonicated
DNA
type
assay
DNA
I was
was
identical
was
0.5
added
mg/mI
to
to
the
agar.
that
of the
0.25%
Five
PDP
agar
assay
solution.
hundred
with
the
The
final
nuclei
were
counted
Giemsa.
and
exception
concentrato
de-
L.I.
slides
of the
transformed
considered
cells.
lymphocyte
All
cells
cultures
other
than
were
stained
nonbasophilic
with
small
lymphocytes
1000
cells
and
to be transformed.
1.
PDP-labeled
nuclei at a final
nification of 1920 x.
lymmag-
were
pyknotic
S
Fig.
phocyte
at
1 : I dilution
Index
Methanol-fixed
were
ml
of labeled
that
assayed
the
deoxythymidine-5’-triphosphate
Five
Labeling
termine
each
l0
8.0
of the
technique.5
DDDP
to
tritiated
of 10
appearance
cells
(pH
of
Schwarz/Mann)
original
were
500
agar
deoxyadenosine-5’-triphosphate,
4 Ci
concentration
of the
ring
x
washed
in 0.25%
leaving
modification
buffer
and
counting
and
radioautographed
labeled.
dipped
membrane,
8.3
and
Tris-HCI
SA
and
of exposure,
by
considered
were
a glass
contained
phosphate,
3 wk
centrifuged,
smears
cytoplasmic
subjected
medium
washed,
Index
suspended,
unfixed
the
After
were
McCoy’s
smeared,
determined
or more
were
The
harvested,
was
Labeling
PDP
M
were
emulsion.
index
four
I Ci
Cells
liquid
labeling
with
Index
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102
SCHIFFER
e
ET
AL.
DODP
o POP
#{163}
‘HTdJ
Fig. 2.
Time relationship
of
3HTdR
PDP, and DDDP labeling indices
in nuclei
of lymphocytescultured
in the presence
or absence
of
phytohemagglutinin.
a
.
.
.
3HTdR
0--,
PDP;
I-,
DDDP.
Flagged
sent 1
paints
SEM of five
or
repremore
values.
RESULTS
The
lation
PDP
value
time
relationship
between
the
radioautographic
L.I.s
after
PHA
stimu-
is shown
in the upper
portion
of Fig. 2. The mean
value
for both
the
and the DDDP
L.I. in five normal
individuals
is 0.04 at time
zero.
This
will be used as a baseline
for the regression
lines
reported
below.
Both
the PDP and DDDP
L.I.s increase
at the same
rate to 0.30 by the second
values
then
diverge
with
the DDDP
LI.
nearly
50%
greater
than
the
value
at the third
and fourth
day.
The 3HTdR
L.I. is consistently
less
either
of the
By
other
measures.
comparison,
stimulated
the
values
lymphocyte
appears
to
be
by day 4.
The linear
day;
PDP
than
for
cultures
a slow
the
are
increase
in all
least-mean-square
DDDP,
shown
three
regression
PDP,
in the
values,
line
and
lower
3HTdR
portion
but
of the
they
PDP
L.I.s
in
of Fig.
never
un-
2. There
exceed
values
between
0.10
days
1-3 (Y = 19.38X
- 9.15)
intersects
the 0.04 baseline
at 16.8 hr and the 0 baseline at I 1 .2 hr. The regression
line for the DDDP
index
( V = 27.57X - 15.43)
intersects
the 0.04 baseline
also at 16.8 hr and the 0 baseline
at 13.6 hr. Depending
appearance
The
from
from
the
upon
transforming
index
day
0 to 4. These
the
PDP
DDDP
During
labeling
which
baseline
is used, these
ofcells
labeled
with 3HTdR.
L.I.
the
smears,
and 0.0085,
technique,
the
PDP
and
more
gradual,
5-1 1 hr prior
lymphoid
exception
cells
of day
transforming
1, are
index
to the
was
initial
measured
indistinguishable
is 0.25,
compared
to
of 0.10.
after
examined
respectively.
the L.I.s
the
mean
PDP
hr
are
PHA-stimulated
with
day-l
the
26
were
blood
A second,
and
initial
indices
0.014
aration
2 hr.
The
of 0.16
of
values,
times
introducing
at short
DDDP
PHA
intervals
labeling
into
(Fig.
indices
for
the
3).
culture,
In
direct
the
mononuclear
cells
After
these
samples
were processed
by
rose to 0.04 and thereafter
to an apparent
increase
began
between
14 and
18 hr.
three
peripheral
were
the seppeak
at
Regression
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HUMAN
103
LYMPHOCYTES
#{149}
DDDP
o PDP
.
HTdR
6
0.2
C
Fig. 3.
Early
time
ship of 3HTdR,
labeling
relation-
PDP, and DDDP
indices
phlebetomy
paint)
separation
stimula-
tion
paints
Flagged
SEM
of
(0-hr
point).
represent
1
five or more values.
paints are individual
and
show
the
Unflagged
values.
a similar
onset
j
and
phytohemagglutinin
lines
0.1
after
( -4-hr
lymphocyte
0
5-1
1-hr
ofincreased
lag
between
3HTdR
the
rise
HOURS
AFTER
in the
PDP
PHA
and
DDDP
L.I.s
labeling.
DISCUSSION
The present
lymphocytes
tion
studies
stimulated
of a short
0,
period,
tion ofcells
in cycle
it appears
that about
cycle.
After
this fraction
serve
with
to define
the
PHA.
Using
the
PDP
assay
is considered
at any particular
time.
4% of the lymphocytes
an acute
rise to 10%,
does not significantly
cycling
characteristics
the previously
mentioned
Following
initially
a measure
thought
the
to
PDP
index
represent
Between
the start
PDP and DDDP
fraction
of
of cells
nuclei
contain
may be daughter
back into cycle
nuclear
DNA
polymerase
but
frac-
DDDP
DNA
point,
2 days,
in
during
which
is followed
by
the maximUm
is
is in cycle at the
the
contain
2-day
after
with the DDDP
L.I. significantly
greater
than
that both
measures
detect
the same
fraction
interval.
In the later time periods,
the increased
cells
gone
in cycle,
which
of the culture
period
and the
L.I.s
are similar.
However,
cells
of the
there
is a 12-16-hr
lag phase
change.
This quiescent
phase
is a measure
the
cultured
assump-
the initial
cell separation,
entered
into culture
are
a progressive
increase
in the fraction
of actively
cycling
cells;
reached
on day 4. By this measure,
52% of the cell population
time.
Whereas
of
index
is
polymerase.
values
for
the values
both
the
diverge
the PDP
value.
Thus,
it appears
of cells during
the initial
2-day
DDDP
L.I. suggests
that some
lack
primer-template
ability.
cells of the original
lymphocyte
division
which
but have retained
some of the previously
synthesized
These
have
not
DNA
polymerase.
These
data may
sion, or availability,
in cycle.
On
the
simultaneously,
to be a general
either
the
other
hand,
biologic
template
the
albeit
at different
phenomenon
in
nuclear
be an initiating
In support
have further
of primer
DNA
polymerase
meaning
in that
may determine
DDDP
and
PDP
they imply
that represwhether
a cell remains
L.I.s
appear
to
rates,
after
PHA
stimulation.
cells stimulated
out of the
or
step into cycle or the first
of this concept
it has been
the
priming
ability
rise
If this
noncycling
of the
DNA
synthetic
process.
shown,
for example,
that
almost
proves
state,
DNA
noncycling
may
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
104
SCHIFFER
cells,
when
tivity
and
stimulated
that
showing
into
this
this
change
are
isolated
PHA-stimulated
plate
activity
within
template
cells
The presence
in cycle.
DNA
WI-38
glands.’5
human
after
lymphocyte
stimulation.’6
in stationary
PDP
phase
L.I.
at 24
plained
the
go
the
probably
equivalence
L.I.;
this
this
imparts
that
4%
cell
population
In examining
PDP
This
and
may
cell
the
DDDP
be due
of the
separation.
compatible
been
found
ternatively,
PHA
lymphocytes
these
The
if the
The
may
is out
cell
following
cycle
1.
PDP
DNA
synthesis
in vitro.
3.
Tc
data
index
=
positivity.
time
assumptions
2.
GF
to justify
both
in
PDP
equals
x Ts/L.I.
using
phase,
02
(Tc)
are
curve
The
of
the
cells
of
single
index
is
as an
that
increase
is in cycle,
procedure,
the
only
l%-2%
we can
conclude
for
a small
periphof the
that
and
either
the
in both
takes
indices.’92’
Heparin
DNA
on
PDP
the
and
is that
and
by
uv irradiation
nuclear
the
peak
by
the
6-hr
period
after
phlebotomy.
process
which
was initiated
in labeling
effect
in
the
by
place
has
polymerase.22
DNA
Al-
synthetic
DDDP
indices
a small
fraction
separation
and
mechincrease
of quiescent
stimulation
cultured
lymphocytes
can
be
lymphocytes
equals
approximately
of
calculated
made:
growth
time
this
x-ray
possibility
of cycle
generally
daughter
population
in culture,
after
a specific
the
are
the
some
is recognized
enrichment
show
From
increase
that
cells
lymphocytes.
the transforming
lymphocyte
endogenous
A third
in
of being
systems
monocytes
have
been
eliminated
has stimulated
a positive
reaction.
repair
this
it appears
activate
median
that
a cell
culture
follows
that
cell
occurring
evoke
is involved.
cells
noted
in the first
of a DNA
repair
activate
could
although
before
events
with
to
PHA
anism,23
initial
It is known
in a period
also
is in cycle.
indices
was
to activation
is observed
is 2.5 times
greater.
This
can be cxincreases
rapidly
after
PHA
stimula-
to the
lymphoid
cells
or
the procedure
itself
cycle,
these
appear
as nontransformed
of the PDP
index
with
basophilia
index.
it is apparent
some
noncycling
procedure
and/or
of
although
definition
of the term,
at the end of the
blood
smears
from
the same
individuals
mononuclear
out
indicates
index
production
and
that
not be representaphenomenon,
that
does not assure
in some
tissue
tissue,
liver,’4
shown
of growth.’3
transformed
DDDP
been
acsystems
also have
increased
temstudies
involved
template
therefore
may
The opposite
phase
hepatic
where
the transforming
by the fact that RNA
tion’8 and
transforming
Although
our
eral
in adult
it has
nuclei
These
cells
the
regenerating
In addition,
polymerase
apparently
has been
observed
and
than
division
to the
hr,
a stationary
of cycle.’7
of morphologically
rather
the first
exception
when
go into
template
Among
fibroblasts,’3
disappearing
that
in chromatin
stimulation.
RNA
synthesis
and
for DNA
synthesis.
of DNA
polymerase
considered
out
The incidence
increase
after
salivary
2 hr
activity
culture
an
early
cultured
activity
for DNA-directed
tive of template
activity
tissue
show
increases
isoproterenol-stimulated
of
cycle,
activity
ET AL.
fraction.
(Ts)
of
10
hr
(observed).
the
PDP
index
in cultured
human
lymphocytes
as an
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
HUMAN
LYMPHOCYTES
Table
Time
105
1. Ca lculated
After
PHA
Cytokinetic
uesfor
Val
Fraction
Growth
(hr)
LI.
Cultured
H uman
(Observed)
Lymphocytes
Estimated G1 (hr)
Tc (hr)
A
24
0.100*
0.015
66.7
48
0.300*
0.215
14.0
72
0.485*
0.325
14.9
1.9
96
0.515*
0.325
15.8
2.8
53.7
1.0
B
*PDP
t
24
0.150t
0.015
100.0
48
0.320t
0.215
14.8
1.8
72
0.705t
0.325
21.6
8.6
96
0.735t
0.325
22.6
9.6
index.
DDDP
index.
estimator
short
of GF
G,
derive
periods,8
from
where
Several
in vitro
ods have shown
as described
our
GF
experience
was
in other
determined
in the
in vivo,
in vitro
The L.I. (observed)
Materials
and
Methods
for the time of 02
If one considers
+ M to be about
3 hr.9
all cells that contain
DNA
of the
primer
proliferative
as previously
that
a long
and
this
While
0 1 period
would
cell
does
preclude
it might
ofhuman
certain
numbers,
be attractive
of unstimulated
factors
viability,
cells,
mittedly
in lymphocyte
that only a relatively
jority
ofgrowth.
We
let
this
noted
Cells
etc.
are
to evolve
are
not
our
required.
initial
If one
data
One
fraction
utilizes
GF
are
obtained
are
with
at this
the
knowledge
the
growth
growth
stimulable
from
time,
values.
for
cell
have
com-
as estimates
cytokinetic
model
of potentially
data
only
to estimate
to
compatible
distributed
of their
must
is a
cells
(some
from
this
estimate,
also
be regarded
a comprehensive
using
(3)
positive)
daughter
data resulting
randomly
has
index
are seen in Table
1,
a reasonable
figure
as the
and
determination
in culture
other
cell
exist.
an accurate
lymphocytes
We shall
the factors
PDP
the few results
found
in the literature
in lymphoblasts
in vivo, in vitro.26
results
in both
parts
A and B must
method
labeling
(DDDP
including
cytokinetic
methvalue
assumption
polymerase
assumption
are seen in Table
1, part B.
The results
presented
for Tc and 0, , using
patible
with
the Tc found
The 24-hr
and
Our results
for this system
for the 0 1 period
assuming
pool,
presumably
discussed),
the
with
techniques.
the double-labeling
is the 3HTdR
pulse
section,
formulation.
an estimate
systems
cytokinetic
Ts by the labeled-mitoses
and, in addition,
a similar
by
classic
cytokinetic
part A, along
with
be part
without
proliferating
by classic
studies
of human
lymphocyte
that the 10-hr value is valid,9”0
for human
lymphocytes
recently
been published.24
eters,
87.0
paramof
cells,
literature,
total
decay
ad-
cultures
prepared
in different
ways,
it appears
probable
small
population
of initial
cells is responsible
for the mahave estimated
this fraction
to be in the range
of 10-20%.
remain
above.
speculation,
however,
since
our
data
do
not
It has been shown
in some systems
using isolation
techniques
and classic
tube assays
employing
exogenous
primertemplateL2.4
that nuclear
DNA
merase
activity
is present
in cells at all stages
of the cell cycle.
However,
include
testpolycyto-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
106
SCHIFFER
plasmic
DNA
synthesis.
polymerase
This
presumed
to take
noted,
in addition
priming
activity
information
increasesjust
is apparently
before,
condition
capable
strip
the
of
acting
as
template
the
than
in the
tion.
Therefore,
these
lymphocytes
Finally,
nificantly
background
PDP
grains
assay,
we consider
the
of,
DNA
synthesis
is
the
DNA
induced
out
of
in the
DNA
synthesis.
radioautographs
polymerase
greater
nuclear
presence
that
in
in loca-
we
is found
of DNA
detect
to increase
synthesis.
studies
by Loeb et al.27 by chemical
lymphocytes
and
are consistent
or
fact,
in
in character.
polymerase
to the start
quiescence28
In
are
is overwhelmingly
nuclear
is that
DNA
5-11
hr prior
are similar
to, and confirm,
of the enzyme
activity
in
for
As
in
cells (as shown
by several
staining
cytoplasmic
DNA
polymerase
is
label
to be predominantly
our evidence
in lymphocytes
systems
time
DNA
DNA
polymerase
activity
actually
when
the cells’ own DNA
is utilized
the slides
in our assays
appears
to
cytoplasm
from
isolated
we feel that
little
or no
Although
DDDP
data
tion
the
and
present.
since
place
within
the nucleus
in the presence
of a polymerase.
to the presence
of the enzyme,
the reaction
requires
DNA
some
studies
have
shown
that
the nuclear
does increase
at the time of DNA
synthesis
as primer-template.4
The agar
coating
of
effectively
techniques),
or at the
paradoxical,
AL.
ET
in synchronized
sigThese
determinawith
data
mammalian
in
cell
cul-
tures.4’29
ACKNOWLEDGMENT
We
should
like
to acknowledge
the
able
technical
assistance
of
Dolores
Migliorato
and
Barbara
Diletusso.
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1974 44: 99-107
Cycling Characteristics of Human Lymphocytes In Vitro
Lewis M. Schiffer, Arnold M. Markoe, Alan Winkelstein, Janet S. R. Nelson and John M. Mikulla
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