(CANCER RESEARCH 36, 1748-1753, May 1976]
In Vitro Labeling and Gold Activation Autoradiography for
Determination of Labeling Index and DNA Synthesis Times
of Solid Tumors'
P. G. Braunschweiger, L. Poulakos, and L. M. Schiffer
CancerResearchUnit, Divisionof RadiationOncology,AlleghenyGeneralHospital,Pittsburgh,Pennsylvania15212
SUMMARY
As a result of these limitations, in vitro methodologies
have been developed whereby cell kinetic information for
In vitro labeling and gold activation autoradiography were individual solid tumors can be obtained. Studies utilizing
oxygen-enriched atmospheres (4) or hyperbaric oxygen
used to determine the [3H]thymidine ([3HJTdR)-Iabeling mdi
conditions (5, 8, 10) to facilitate the uptake of [3H]TdR into
ces and DNA synthesis times for C3H/He spontaneous
tissue fragments have been relatively successful. Livingston
mammary tumors.
Three variations of the [3H]TdR, [‘4C]thymidine([‘4C]TdR) et a!. (17) recently reported a method utilizing the Hypaque
Ficoll system described by Boyum (2) and high specific
double-labeling method , together with double-emulsion au
tomadiognaphy, were used to determine the DNA synthesis activity [3H]TdR to rapidly determine Lb's for human mela
times ([email protected] labeled totally in vivo (in vivo-in vivo noma and lung cancer from single-cell suspensions labeled
in vitro.
method) and tumors labeled with [3H]TdR in vivo and subse
The [3C]TdR, [‘4C]TdR
double-label technique (36) for the
quently labeled with [‘4C]TdR
in vitro showed similar T8
values. DNA synthesis times for tumors determined totally in determination of the T@has been adapted for use in human
tumors by administering the 2nd label, [‘4C]TdR,
in vitro to
vitro by double labeling (in vitro-in vitro method) were sig
nificantly longer than those observed in vivo; however, disaggregated cells from tumors labeled with [3H]TdR in
identical samples subjected to Hypaque-Ficoll gradient sep
situ. This in vivo-in vitro method has proven useful in cell
anation after double labeling showed T8's similar to those kinetic studies of bone marrow cells (15, 36), spleen cells
found in vivo. Furthermore, the interval between [3H]TdR (18, 31), ascitestumors(15), lymphocytes(24, 31), leuke
and [‘4C]TdR
administration had no effect on T@estimates in mias (12, 36), and melanoma cells in the bone marrow (11).
Although the T@values determined by this method for these
vitro. Gold activation automadiogmaphy was used in the pres
easily dissociable tumors are in good agreement with values
ent experiments to reduce automadiognaphic exposure
times. This method, together with in vitro labeling, permits
found by in vivo methods, automadiogmaphicexposure times
[3H]TdR labeling index and T8determinations after 6-hr and of up to 6 weeks are often required.
Fabrikant (7, 8) and Fabnikant and Cherry (9), utilizing
7-day exposures, respectively.
hyperbaric oxygen to facilitate the uptake of [3H]TdR and
[‘4CITdR
into tissue fragments in vitro, devised a totally in
INTRODUCTION
vitro double-labeling method to measure Ta's for human
solid tumors and normal tissues (in vitro-in vitro method).
Since many therapeutic modalities currently used in can
Although the results using this method correlate well with
cer therapy exert their effect during specific phases of the other studies, the method is time consuming in that paraffin
cell cycle, cell kinetic information on tumor cell populations
sections and long autoradiographic exposure times are me
may be important for the future design of treatment proto
quired. Others, using the in vitro-in vitro method at ambient
cobs. The percentage labeled mitoses method has yielded
conditions, have measured Ta's for easily dissociable tis
valuable information on a variety of human solid tumors;
sues such as tonsillam lymphocytes (32), bone marrow cells
however, it is limited in application to situations where
(14, 16), and melanoma cells in bone marrow (11). Although
multiple biopsies are feasible. In addition, in vivo adminis
in vitro-in vitro T8's for these cells types were in good agree
tration of [3H]TdR2 may not only be a potential hazard in ment with results obtained with other methods, equivocal
patients with good prognosis but may also preclude follow
findings have been observed (35) with this method for hu
up on serial studies throughout treatment. Furthermore, the man breast cancer.
long autoradiognaphic exposure times required in these
In attempts to reduce automadiographic exposure times,
studies menderthe results of little on no benefit in planning
methods to improve the efficiency of both the automadi
the course of therapy for the patient.
ognaphic procedure and the developing process have been
devised. Panayi and Neill (26) and others (1, 6) exposed
autoradiogmams in liquid scintillation counting fluids, effec
NO1-CB-43899 and in part by Clinical Radiation Therapy Research Center
Grant CA-i 0438.
tiveby decreasing the exposure times. At the electron micro
2 Abbreviations
used
are:
TdR,
thymidine;
LI, labeling
index;
NARG,
nor
scope level, gold salts have been used to intensify the latent
mal autoradiography; GARG, gold activation autoradiography.
image in sensitive emulsions (29, 30). Rechenmann et a!.
Received August 6, 1975; accepted February 6, 1976.
I These
1748
studies
were
supported
by
Breast
Cancer
Task
Force
Contract
CANCER
RESEARCH
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VOL.
36
DNA Synthesis Time Determinations in Vitro
(27, 28) reported a similar method to increase the efficiency
of developers and better resolve nuclear tracks resulting
from ‘4C
/3 emissions (38).
Prior to our initiation of studies with human tumors, the
feasibility of performing LI and T@measurements, totally in
vitro, was explored in the spontaneous mammary tumor of
the C3H/He retired breeder. It was also felt that a rigorous
comparison of in vitro with in vivo measurements should be
performed.
In the present studies, the in vitro labeling method of
Livingston et a!. (17) was used and modified to determine
the [3H]TdR LI and the T,@by [3HJTdR, [‘4C]TdR
double
labeling and double-emulsion automadiography. Further
more, an improved automadiogmaphictechnique, using acti
vation ofthe nucleantrackemulsion, was used in an attempt
to reduce exposure times to within a clinically useful mange.
cium-free Eagle's minimal essential medium (Grand Island,
Biological Co.). This medium was used to minimize the
formation of cell aggregates. In some experiments, drop
preparations were made immediately after the labeling pe
nod; however, in most experiments, the suspensions were
applied to Hypaque-Ficoll gradients (2) and centrifuged at 4°
for 20 mm at 400 x g. Cells confined to a narrow band at the
gradient-media interface were carefully pipetted off and
diluted
in7 to8 volumesoffreshmedia.The viability
deter
mined by trypan blue exclusion in postgmadient samples
was consistently greater than 80%, while only 20% or less of
the cells were trypan negative in pme-gradient samples. The
cells at the gradient-media intemphase were primarily large
mononuclear cells, while cells at the bottom of the gradient
consisted primarily of polymomphonuclear leukocytes,
enythmocytes, and tnypan-positive mononuclear cells. Only
2% or less of the cells in this fraction were tnypan negative.
Drop preparations of the cells collected from the gradient
MATERIALS AND METHODS
intemphase were prepared as described above.
In Vivo-in Vitro Double Labeling. Tumor-bearing animals
[3H]TdR and sacrificed
Tumor System. Tumor-beamingC3H/He (JAX) mice were were given i.p. injections of 40 @Ci
housed 4 to 6 animals per cage, fed standard mouse chow 60 mm later. Single cell suspensions were prepared as
(Ralston Purina Company, St. Louis, Mo.) and water ad described above, and cells were incubated for 30 mm at 37°
libitum. Tumors were measured in 3 dimensions twice
with [‘4C]TdR
(0.25 @Ci/ml).The reactions were terminated
weekly with calipers to determine the volumetric growth
on ice and the suspensions were applied to Hypaque-Ficoll
gradients and centrifuged for 20 mm at 400 x g at 4°.Drop
characteristics for each tumor. The mean volume for tumors
used in these studies was 1.26 ±0.13 cu cm.
preparations from cells collected from the interface of the
In Vivo Labeling. In single-label studies, tumor-beaming gradient were made as described above.
animals were labeled for 1 hr with a single i.p. injection of
Autoradiography. In all studies, Kodak NTB-2 liquid
40 @Ci
of [3HITdR (14 to 17 Ci/mmole; Amersham/Seamle, emulsion was applied by dipping at 45°.The autoradi
Arlington, Heights, Ill.). In double-label studies, similarly
ogmams were dried at room temperature for 1 hr and ex
treated animals were labeled for an additional 30 mm with posed in light-tight boxes at 4°for from 6 hr to 30 days.
2.5 @Ci
of [4C]TdR (54@Ci/mmobe; Amersham/Seambe). Im
NARG (utilizing conventional developing techniques) and
mediately following the labeling periods, the animals were GARG were used in these experiments. In the latter, a
sacrificed by cervical dislocation and the tumors were ex
solution of gold salts (13) was used to activate the latent
image produced in the photographic emulsion prior to de
cised and placed on ice.
The tumors were minced with fine scissors in McCoy's vebopment. The gold solution was prepared by dissolving
medium with 20% fetal calf serum (Grand Island Biological
75.6 mg KAuCI4 (Vamlacoid Chemical Co., Elizabeth, N. J.)
Co., Grand Island, N.Y.). Tissue fragments were further
and 100 mg KSCN in 20 ml of triple-distilled water (Fisher
disaggregated with a Pasteur pipet and the suspension was Scientific, Pittsburgh, Pa.). This solution was then heated to
filtered through a stainless steel screen. The cells were 5°and 300 mg KBmwere added. When the solution had
cooled, the volume was brought to 500 ml with tniple-dis
washed in fresh medium and resuspended in 0.1 M citric
acid for 1 mm at 4°.Drop preparations (35) were made by tilled water. Since the shelf life of this solution is very short,
nesuspending the cells in Clarke's fixative for 5 mm at 4°, it was prepared just before use to ensure greatest activity
and lowest. background.
and then applying 1 on 2 drops of the suspension to acid
cleaned slides. The slides were air dried, washed for 20 mm
Automadiogmamsprocessed by the NARG procedure were
in running tap water, then washed briefly in 3 changes of developed in Kodak D-19 for 10 mm at 15°,fixed in acid fixer
triple-distilled
waterand airdriedina dust-free
area.
(10 mm), washed in tap water (30 mm), and air dried. Auto
In Vitro Labeling. Single-cell suspensions for in vitro sin
nadiogmamsprocessed by the GARG procedure were incu
bated in 3 changes of triple distilled water and developed at
gle and double labeling were prepared in a manner similar
The slides
were fixed,
to that described above. Tumors were minced with fine 15° in Kodak amidol developer.
scissors in McCoy's medium with 20% fetal calf serum at washed, and aimdried as in the NARG method. For both
room temperature, the suspension was filtered, and the methods the developed automadiogmamswere stained with
cells were resuspended (3 to 5 x 10@cells/mI) in fresh Harris hematoxylin. In single-label studies, the LI was deter
media. Three-mI aliquots were incubated at 37°with 2.5 j.tCi mined by counting at least 1000 cells/tumor.
[3HJTdRpen ml for 60 mm. In double-label experiments, the
Double-label samples were subjected to double-emulsion
cells were labeled for an additional 30 mm with [‘4CITdR
at a autoradiogmaphy since it has been shown to be the most
final concentration of 0.25 MCi/mI. At the end of the labeling accurate autoradiognaphic method for distinguishing be
period, the reactions were stopped on ice, the suspension tween cells labeled with only [3H]TdR and cells labeled with
was centrifuged, and the cells were resuspended in cab [‘4C]TdR
(12). In these studies, the 1st and 2nd emulsion
MAY 1976
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1749
p.
G.
Braunschweiger
et
a!.
layers were separated by a thin layer of celloidin (5% par
loidin in ethem:alcohol, 1:1) applied by dipping. This layer
not only protects the staining of the cells during subsequent
steps, but also prevents the weaker tritium p particle from
penetrating into the 2nd emulsion layer. The 2nd emulsion
was then applied, exposed and developed (NARG onGARG)
like the first. The T5, calculated from the equation of Wimber
and Quastler (37)
Table 1
(3HJTdRLi's for C3Hspontaneousmammarytumors in vitro with
GARG
(%)NARG
TumorLI
hr)1618.06.88.11627.15.57.416315.812.712.516414.112.614.51656.76.87.01666.46.37.016
(3-5 days)GARG (24 hr)GARG (6
T8 = [‘4C/3H]TA
where ‘4C
is the number of cells labeled in both emulsion
layers, 3H is the number of cells labeled in only the bottom
layer and TA is the interval (hours) between administration
of the isotopes ([3H]TdR and [‘4CJTdR).
This result was
determined by examining at least 500 labeled cells for each
tumor. The relatively few peripheral blood cells (mostly
small lymphocytes) in these preparations were easily recog
nized momphobogically and disregarded.
Background in both single- and double-emulsion autora
diogmams was consistently less than 2 grains cell, regard
less of the developing method (NARG on GARG) used, and
cells labeled with 10 on more grains per cell were consid
ered positively labeled. In mouse in vitro experiments, how
even, grains over labeled cells were generally too numerous
to count. Double-emulsion autoradiognarns of cells labeled
with only [3H]TdR showed essentially background labeling
in the 2nd emulsion regardless of the method (NARG on
GARG) or exposure time (between 6 and 30 days) used.
±SE.7.8
0.6Mean
±S.E.b7.1
a ND,
(.
@
Table 1 shows the in vitro [3H]TdR Lb's for 23 consecu
tively studied C3H/He spontaneous mammary tumors. Sam
pIes from the same labeled cell suspension for each tumor
were subjected to both the NARG and GARG procedures. In
addition, samples from most tumors were subjected to
GARG analysis after both a 6-hr and 1-day exposure period.
In this study in vitro, Lb's ranged from 15.8 to 4.2%. In vitro
Lb's for most tumors, obtained by GARG procedure, were
similar to those obtained by the NARG method. The conmela
tion coefficients for NARG versus GARG (24 hm)and NARG
versus GARG (6 hr) were 0.703 and 0.681 , respectively, both
values representing highly significant correlations (p
0.01). Grains pen labeled cell were too numerous to count in
all samples except GARG (6 hr), where approximately 30 to
40 grains per labeled cell were noted.
Also shown in Table 1 is the mean in vivo LI for 24
consecutively studied C3H/He spontaneous mammary tu
mors. Although in vivo and in vitro Lb'swere not determined
for the same tumors, in these studies the group LI's in vitro
were not different from those found in vivo (Student's t test
and Wilcoxon test for unpaired data).
The overall results comparing the 3 double-label methods
for determination of the T5 for C3H/He spontaneous mam
many tumors are shown in Table 2. A total of 51 sponta
neous tumors were studied. In vitro double labeling me
suIted in a mean T8value that was similar to values found by
1750
±
±0.67.7
±0.7
(24@
b In
RESULTS
±0.78.1
not
done.
vivo.
Number
in
parentheses,
number
of
consecutive
tumors
labeled
in vivo.
2T,'s
Table
for C3H/Hespontaneousmammarytumors determined by 3
double label techniques and double emulsion
autoradiographyT@
(hr)Method
(hr)In
Mean ±S.E.
vivo-in
vivo
In vitro-in vitro
In vivo-in vitro
a Numbers
Range
TA
10.7±0.2(16)°
12.3-9.2
1.0
10.9 ±0.2 (26)
10.5 ±0.2 (9)
12.5-9.5
11.7-9.8
1.0
1.0
in parentheses,
number
of tumors.
double labeling totally in vivo by the vivo-vitro double-label
ing method. Furthermore, the range of T@values were quite
similar for all 3 groups.
Twelve of the 26 tumors studied totally in vitro and me
ported in Table 2 were not only autoradiogrammed by the
NARG method, but also with the GARG technique. In addi
tion, T8values for these tumors were determined from dou
ble-babeled cell suspensions prior to Hypaque-Ficoll gra
dient separation. The results (Table 3) indicate that, al
though automadiographic exposure times were reduced to
within 1 week, using the GARG method, themewas no signif
icant difference between T8 values for the 2 autonadi
ogmaphic methods. Samples not subjected to Hypaque-Fi
coIl gradient separation consistently showed longer T@val
ues than those observed for cells following Hypaque-Ficoll
separation.
CANCERRESEARCHVOL. 36
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1976 American Association for Cancer Research.
DNA Synthesis Time Determinations in Vitro
Since cells held in vitro during the labeling period could
undergo changes in the DNA synthesis rate, or changes in
the mateat which cells enter S phase, 2 experiments were
done to test the effect of our culture conditions on DNA
synthesis in C3H/He spontaneous mammary tumor cells. In
the 1st experiment, duplicate cell suspensions were pre
pared from each tumor as described in the ‘
Materials and
Methods.' â€B̃oth were incubated at 37°,one with [3HJTdR
and one without. After 1 hm,the [3HJTdR-Iabeled cells were
harvested, while the duplicate culture was labeled for 30
mm with [‘4C]TdR.
At the end of each labeling period, the
cells were subjected to Hypaque-Ficoll separation and
single- ([3H]TdR cells) or double- ([‘4CJTdR
cells) emulsion
autonadiognaphy (GARG). The results (Table 4) show similar
Lb's for both the [3H]TdR- and [14CJTdR-labeled samples,
suggesting that cells segregated by the Hypaque-Ficoll
gradient continue to enter S phase after 1 hr in vitro at a
matesimilar to that seen for cells labeled immediately.
Table 3
In vitro T,'s for 12 C3Hspontaneousmammarytumors determined
from double emulsion autoradio
gramsT8
Table 5
Thedifferentintervals
T5in vitro for 4 spontaneousC3H/Hetumors using
(Ta) between (3HJTdR andadministrationT@―
f'@CJTdR
(hm)Tumor
1TA
S.E.0.50
(hr)
397
Tumor
398
Tpmor
399
Tumor
340
0.10.67
0.30.84
10.7
9.7
10.7
10.7
10.5
10.9
10.5
10.5
10.6 ±
10.5 ±
10.3
10.5
10.8
10.2
10.9
10.6
10.7 ±
10.2
10.0
10.2±0.3
0.11.00
10.9
10.6±0.21.5011.0
10.9
a DNA
synthesis
time
9.5
(hr)
=
Mean ±
[TA][14C/3H].
In another experiment, to evaluate the mateof DNA synthe
sis during the in vitro labeling period, the interval between
[3H]TdR and [‘4CITdR
was adjusted between 30 and 90 mm.
The results from this study are shown in Table 5. The data
show that the TA interval between 30 and 90 mm had no
significant
effect
on thecalculated
T@values.
DISCUSSION
(hr)Tumor
In the present study, [3H]TdR Lb's and T@values were
determined for C3H/He spontaneous tumors in vivo and in
vitro. In vitro LI and T8 values for this tumor were similar to
10.2
10.5
those observed in vivo and in good agreement with previ
14.7
232
9.9
10.2
14.8
233
12.5
11.6
ously reported values (19-25). However, both in vivo and in
13.5
234
11.0
10.8
vitro Lb'swere in the low range of values reported by Bresci
15.4
241
10.2
11.0
ani (3). This discrepancy may be a result of different sam
13.7
245
10.4
10.9
pling methods, since his Lb's were reflective of cell popuba
16.8
246
9.7
10.5
tions near the cortical shell of the tumor and might not
15.5
247
11.4
11.1
248
10.4
11.2
13.9
reflect the total cell population.
13.1
250
9.6
10.7
In vitro duplication of T@estimates made by double label
15.1
251
11.1
10.8
ing
of tumor cells in situ was, in the present studies, contin
17.6
254
10.7
10.5
gent upon the use of the Hypaque-Ficoll gradient separation
14.8 ±0.4
Mean ±SE.
10.6 ±0.3
10.8 ±0.1
(12)―
(12)―
(12)13.8
technique (2). That others (11, 14, 16, 24, 32, 36) have
demonstrated representative in vitro-in vitro T8's may be due
a Autoradiographic
exposure
times
of 1 day and 21 days
for the
in part to the nature of the tissues studied. Since bone
1st and 2nd emulsion, respectively.
b Exposure
times
of 1 day
6 days
for
the
1st
and
2nd
emulsion,
marrow cells, peripheral blood cells, and lymphoid tissue
respectively.
can be easily disaggregated, suspensions with high viability
C Sample
not
subjected
to
Hypaque-Ficoll
separation
or
NARG
and few damaged cells can be easily prepared. In the pres
procedure.
ent experiments, where extensive mincing was necessary
d Numbers
in parentheses,
number
of tumors.
for adequate disaggmegation, only 20 to 30% of the cells
cells.
were tmypannegative prior to the labeling periods. That the
postgnadient viabilities were consistently in excess of 80%
Table 4
and the total number of trypan-negative cells recovered
[3HJTdRand('4CJTdRLI's for C3Hspontaneousmammarytumors in from the gradients was usually about one-third of the total
vitro
trypan-negative cells applied would suggest that, aside
(%)[3H]TdR―[‘4C]TdRb2984.95.22997.66.63018.86.93025.57.53058.96.73066.76.5Mean
from nonviable cells, damaged cells possibly synthesizing
DNA at subnormal rates were also removed. Thus in the
TumorLI
present studies as in previous vitro-vitro studies, T@determi
nations made on samples relatively free of damaged and
nonviable cells are the most reliable.
The finding that the [3H]TdR Lb'sfor cells during the 1st 60
mm in vitro were similar to that for cells incubated at 37°for
1 hr prior to [‘4C]TdR
would suggest that the cells separated
±S.E.7.0
±0.76.5
±0.4
by the Hypaque-Ficoll gradients are progressing from G1
into DNA synthesis at or near normal mates. Furthermore,
a One
hr label.
the calculated DNA synthesis times were similar for all TA'S,
b One
hr preincubation,
0.5-hr
label.
GARG―Pregradientc231
NARG―
@
V
Significantly
longer
(p
0.1)
than
Hypaque-Ficoll-separated
MAY 1976
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1751
P. G. Braunschweiger et a!.
between 30 and 90 mm, suggestingthatinHypaque-Ficoll Tumors with Tritiated Thymidine. Cell Tissue Kinet., 6: 217-227, 1973.
6. Dune, B. , and Salmon, S. High-speed Scintillation Autoradiography
segregated cells the mateat which cells complete DNA syn
(HSARG) for Clinical and Research Hematology (Abstract). Blood, 44:
thesis is also constant during the labeling period.
914, 1974.
7. Fabrikant, J. I. The Kinetics of Cellular Proliferation in Human Tissues.
Although cell suspensions obtained after Hypaque-Ficoll
Determination of Duration of DNA Synthesis Using Double Labeling
gradient separation are not totally devoid of nontumon cells
Autoradiography. Brit. J. Cancer, 24: 122-127, 1970.
8. Fabrikant, J. I. The Kinetics of Cellular Proliferation in Normal and
(lymphocytes and macrop@ages), the similarity between the
Malignant Tissues: A Review of Methodology and the Analysis of Cell
results form in vitro and in vivo labeling studies suggests
Population Kinetics in Human Tissues. Am. J. Roentgenol. Radium Ther
that the cell population isolated from the Hypaque-Ficoll
apyNucl. Med., 111: 700—711,
1971.
9. Fabrikant, J. I., and Cherry, J. The Kinetics of Cellular Proliferation in
gradients were reflective of the cell population labeled in
Normal and Malignant Tissues. x. Cell Proliferation in the Nose and
vivo. Furthermore, other studies (unpublished data) from
Adjoining Cavities. Ann. Otol. Rhinol. Laryngol.. 79: 572-578, 1970.
this laboratory using the 13762 transplantable matmammary 10. Fabrikant, J., Wisseman, C., and vitak, M. The Kinetics of Cellular
Proliferation in Normal and Malignant Tissues. II. An in Vitro Method for
tumor, a tumor line with a relatively high [3H]TdR Lb (ap
Incorporation of Tritiated Thymidine in Human Tissues. Radiology, 92:
proximately 30%) and a short DNA synthesis time (approxi
1309-1320, 1969.
mately 6 hr) have also shown good agreement between LI 11. Hagemann, R. F., And Schiffer, L. M. Cell Kinetic Analysis of a Human
Melanoma In Vitro and In Vivo-Vitro. J. NatI. Cancer Inst. . 47: 519-525,
and T5 values determined in vivo and in vitro.
1971.
GARG proved to be an extremely useful tool to shorten
12. Harriss, E. B., and Hoelzer, D. An Evaluation of Various Double Labeling
and Autoradiographic Techniques for the Measurement of DNA Synthe
autoradiognpahic exposure times. Rechenmann and Wit
sisTime in Leukemic Cells.J. Microscopy,96 (Part2): 205-217, 1972.
tendomp (29) found that gold activation could increase grain 13. James, T. The Site of Reaction in Direct Photographic Development. II.
Kinetics of Development Initiated by Gold Nuclei. J. Colloid Sci. . 3: 447density 2- to 3.5-fold, depending on the emulsion type,
455, 1948.
isotope, developer, and developing times. In the present
14. Kesse-Elias, M., Harriss, E. B., and Gyftaki, E. In Vitro Study of DNA
studies, a similar autonadiognaphic enhancement was
Synthesis Time and Cell Cycle Time in Erythrocyte Precursors of Normal
and Thalassemic Subjects Using 3H and ‘@C
Thymidine Double Labeling
noted, although excessive background (>5 grains/cell) was
Technique. Acta Haematol., 38: 170-177, 1967.
observed for developing times greaten than 20 mm.
15. Lala, P. K. Measurement of S Period in Growing Cell Populations by a
Although the autonadiographic sensitivity can be substan
Graphic Analysis of Double Labeling with 3H-and 4C-Thymidine. Exptl.
Cell Res., 50: 459-463, 1968.
tially increased with gold activation, the resolution is me
16. Lala, P. K., Maloney, M. A. , and Patt, H. Measurement of DNA Synthesis
duced. This is a result of the increased size and close
Time in Myeloid-Erythroid Precursors. Exptl. Cell Res., 38: 626-631,
1965.
proximity of the developed silver grains. In studies where
17. Livingston, R., Ambus, U., George, S.. Freireich, E. J., and Hart, J. In
grain counting or definitive localization of labeled com
Vitro Determination of Thymidine-3H Labeling Index in Human Solid
pounds is of primary interest, emulsions diluted 1:1 with
Tumors. Cancer Res., 34: 1376-1380, 1974.
18. Markoe, A., OKunewick, J., and Schiffer, L. M. Kinetic Analysis of
distilled water may be used with somewhat better resolution
Splenic Erythropoiesis in Mice Under Prolonged Hypoxic Stress. Exptl.
and only slightly less autonadiognaphic enhancement.
Hematol., 1: 340-349, 1973.
Recently, an in vitro method for the estimation of the 19. Mendelsohn, M. The Kinetics of Tumor Cell Proliferation. In: Cellular
Radiation Biology, The University of Texas M. D. Anderson Hospital and
growth fraction for solid tumors has been reported (33).
Tumor Institute at Houston, pp. 498-513. Baltimore: The Williams &
This method, together with the time-saving features of
Wilkins Co., 1965.
20. Mendelsohn, M. Autoradiographic Analysis of Cell Proliferation in Spon
GARG and the ease and reproducibility of oumin vitro label
taneous Breast Cancer of the C3H Mouse. II. Growth and Survival of
ing techniques, may render cytokinetic analysis of human
Cells Labeled with Tritiated Thymidine. J. NatI. Cancer Inst., 25: 485-500,
tumors a more practical clinical consideration. In addition,
1960.
21. Mendelsohn, M., and Dethlefsen, L. A. Cell Proliferation and Volumetric
these methods may represent a faster and less tedious
Growth of Fast Line, Slow Line and Spontaneous C3H Mammary Tu
approach to the study of solid tumor cytokinetics following
mors. Cancer Res., 27: 61-74, 1967.
perturbations with chemothenapeutic agents on ionizing ma 22. Mendelsohn, M., and Dethlefsen, L. Cell Proliferation and Volumetric
Growth of Fast Line, Slow Line, and Spontaneous C3H Mammary Tu
diations.
ACKNOWLEDGMENTS
The authors would like to thank Linda Fisher, Tamara Hamilton, Debra
lannuzzi, and Kathleen Klueber for their technical assistance and Paula
Banachoski for preparation of the manuscript.
REFERENCES
1. Bertram, J. , and Heidelberger, C. Cell Cycle Dependency on Oncogenic
Transformation Induced by N-Methyl-N'-nitro-N-nitrosoguanidine
in Cul
ture. Cancer Res., 34: 526-537, 1974.
2. Boyum, A. Isolation of Mononuclear Cells and Granulocytes from Human
Blood. Scand. J. Clin. Lab. Invest., 21: 77-89, 1968.
3. Bresciani, F. A Comparison of the Cell Generation Cycle in Normal,
Hyperplastic and Neoplastic Mammary Glands of the C3H Mouse. In:
Cellular Radiation Biology, The University of Texas M. D. Anderson
Hospital and Tumor Institute at Houston, pp. 547-557. Baltimore: The
Williams & Wilkins Co., 1965.
4. Coons, H., Norman, A. , and Nahum, A. In Vitro Measurements of Human
Tumor Growth. Cancer, 19: 1200-1204, 1966.
5. Denekamp, J., and Kallman, R. In Vitro and In Vivo Labeling of Animal
1752
mors. Proc. Am. Assoc. Cancer Res., 9: 47, 1968.
23. Mendelsohn, M. , Dohan, C., and Moore, H. A. Autoradiographic Analysis
of Cell Proliferation in Spontaneous Breast Cancer of C3H Mouse. I.
Typical Cell Cycle and Timing of DNA Synthesis. J. NatI. Cancer Inst. . 25:
477-488, 1960
24. Miller, 5. C., and Osmond, D. G. The Proliferation of Lymphoid Cells in
Guinea Pig Bone Marrow. Cell Tissue Kinet., 6: 259-269, 1973.
25. Nordenskjold, B., Zetterberg, A., and Lowhagen, T. Measurement of
DNA Synthesis by 3H-Thymidine Incorporation into Needle Aspirates
from Human Tumors. Acta Cytol., 18: 215-221, 1974.
26. Panayi, G., and Neill, W. Scintillation Autoradiography. A Rapid Tech
nique.J. Immunol. Methods,2: 115-117, 1972.
27. Rechenmann, A., and Wittendorp, E. High Efficiency Development Pro
cedures for Nuclear Emulsions. J. Microbiol., 96: 227-244, 1972.
28. Rechenmann, R., Wittendorp, E., Gielink, A., and Ringoet, A. Lacalisa
tion du Calcium au Niveau des Chloroplastes de Ia Feville d' Avoine
(Avena Sativa L.) par Une MCthode Trace-Autoradiographique a Tres
Hautirrésolution.Compt. Rend., 269: 1525-1527, 1969.
29. Salpeter, M., and Bachman, L. Autoradiography with the Electron Micro
scope. A Procedure for Improving Resolution, Sensitivity and Contrast.
J. Cell BioI., 22: 469-477, 1964.
30. Salpeter, M., and Szabo, M. Sensitivity in Electron Microscope Autora
diography. I. The Effect of Radiation Dose. J. Histochem. Cytochem. , 20:
425-434, 1972.
31. Schiffer, L. M. Human Lymphocyte Proliferation. DNA Synthesis Time.
Cell Tissue Kinet., 4: 585-595, 1971.
CANCERRESEARCHVOL. 36
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1976 American Association for Cancer Research.
DNA Synthesis Time Determinations in Vitro
32. Schiffer, L. M. Observations on the in Vitro Measurements of Human
Lymphocyte DNA Synthesis Time. Cell Tissue Kinet. , 4: 597-599, 1971.
33. Schiffer, L. M., Markoe, A. M., and Nelson, J. S. R. Evaluation of the PDP
Index as a Monitor of Growth Fraction During Tumor Therapy. In: The
Cell Cycle in Malignancy and Immunity, pp. 459-472. Oak Ridge, Tenn.:
Technical Information Center, U. S. Energy Research and Development
Administration, 1975.
34. Simpson-Herren, L. , and Lloyd, H. Kinetic Parameters and Growth
Curves for Experimental Tumor Systems. Cancer therapy Rept. , 54: (Part
1):143-174, 1970.
35. Silvestrini, R., Sarifilippo, 0. , and Tenesco, G. Kinetics of Human Mam
mary Carcinomas and Their Correlation with the Cancer and Host Char
acteristics. Cancer, 34: 1252-1258, 1974.
36. Vincent, P. C., Cronkite, E. P., Greenberg, M. L., Kirsten, C., Schiffer, L.
M. , and Stryckmans, P. A. Leukocyte Kinetics in Chronic Myeloid Leuke
mia: I. DNA Synthesis Time in Blood and Marrow Myelocytes. Blood, 33:
843-850, 1969.
37, Wimber, D. E., and Quastler, H. A ‘4Cand 3H-Thymidine Double Labeling
Technique in the 5tudy of Cell Proliferation in Tradescantia Root Tips.
Exptl. Cell. Res., 30: 8-22, 1963.
38. Wintryth, E., Wittendorp, E., Ittel, M-E., Rechenmann, R., and Mandel,
P. Mise en Evidence par Trace-Autoradiographie Activéede Ia Synthesis
de DNA Nucleolaire dans Ia Fare de Rat Adulte. Compt. Rend. , 277:
1033-1036, 1973.
MAY 1976
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1976 American Association for Cancer Research.
1753
In Vitro Labeling and Gold Activation Autoradiography for
Determination of Labeling Index and DNA Synthesis Times of
Solid Tumors
P. G. Braunschweiger, L. Poulakos and L. M. Schiffer
Cancer Res 1976;36:1748-1753.
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