[CANCER RESEARCH 28, 898-905,May 1988]
Cell Proliferation and Carcinogenesis in the Hamster Cheek Pouch1
Allan B. Reiskirr 3 and Roger J. Berry
Radiobiology Laboratory, Radiotherapy Department, The Churchill Hospital, Headington,
cal and Medical Research, Argonne National Laboratory, Argonne, Illinois 60439
SUMMARY
Tumors were induced in the cheek pouches of Syrian ham
sters of three strains by the topical application of 9,10-di-
methyl-l,2-benzanthracene. Mean tumor latent periods and
growth rates were determined and tumor cell transit times
were measured by tritiated thymidine labeling. Transit times
were also measured in untreated cheek pouch epithelium of
newborn and adult hamsters and during carcinogen-induced
hyperplasia.
Within each strain there were qualitative correlations be
tween the mean tumor latent period and the subsequent growth
rate of the tumors which developed. Significant differences in
the mean latent periods and growth rates of tumors in the
different strains of hamsters were observed. Interstrain com
parisons indicated that the rate of tumor growth depended
more on the existence of a "growth-fraction" and on other
factors than on tumor cell transit times. There were indications
that nongrowing cells could be divided into two groups: (a)
cells with a permanent proliferative incapacity, and (o) non-
growing cells capable of resuming proliferative activity.
INTRODUCTION
The radiosensitivity and chemosensitivity of mammalian
cells varies through the cell cycle (3, 6, 24-26). Biologists and
therapists need to know more about the proliferation kinetics
of normal and malignant tissues to exploit naturally occurring
differences in sensitivity. Although generally impractical in
clinical situations, it is relatively simple to make kinetic meas
urements using autoradiography and tritiated thymidine la
beling or a variety of other technics. Estimates of prolifera
tive activity made from parameters such as the mitotic index
are unreliable (22, 23), and, although there is no apparent
means of accurately and rapidly mesuring kinetic parameters
in single tissue samples, accumulated quantitative data from
experimental systems continue to indicate new approaches to
therapy.
1 Work supported by the U. S. Atomic Energy Commission.
2 Aided by a grant for a Postdoctoral Research Fellowship from
the American Cancer Society, Inc.
3 Present Address : Division of Biological and Medical Research,
Argonne National Laboratory, 9700 South Cass Avenue, Argonne,
Illinois 60439.
* The Radiobiology Laboratory is supported by a grant from the
Medical Research Council.
Received August 31, 1967; accepted January 11, 1968.
898
Oxford, England,* and Division oj Biologi
This report describes a series of experiments in which
changes in proliferation kinetics were studied during carcino-
genesis and aging in the epithelium of the hamster cheek
pouch. It also examines the relationship of proliferation kinetics
to tumor growth and considers the strain dependence of these
parameters.
The hamster cheek pouch was selected to study this response
for several reasons. It is lined with a stratified squamous epi
thelium which has neither follicles nor glands. This simple
tissue should be less susceptible to cyclic changes than more
complex tissue in which changes are likely to occur as the
result of functional demands on accessory structures. Cheek
pouch epithelium appears to have only one stem cell popula
tion, the basal cells. It behaves as a steady state in the adult
animal. These characteristics allow a more straightforward
kinetic analysis than is possible in skin or other epithelial tis
sues.
The pouch responds vigorously to carcinogens applied top
ically, yielding multiple squamous carcinomas of a rather uni
form histologie type. Since each animal has two pouches, it
provides its own control. The cheek pouch is an immunologically
privileged site which tolerates homografts and, under some
conditions, heterografts (4, 8). Tumors arising in it are not as
likely to initiate the immune reactions reported for chemically
induced tumors in other rodents (20, 27). Its rich blood supply
assures both adequate nourishment and oxygen and this cir
culation is, perhaps, responsible for the tissue's ability to main
tain a constant temperature under conditions of extreme en
vironmental variability (14). The anatomic characteristics of
the pouch afford easy access and observation.
MATERIALS AND METHODS
Three strains of Syrian hamsters (Cricetus auratus) were
used in these experiments (J. K. Stewart, The Cottage, Ashton,
Chester, England). Animals from two strains, designated by
coat color as cream and golden, were obtained by random
breeding. The third strain, dark-eared partial albinos (DEA),
was between the 14th and 23rd generations of sib-matings.
Tumors were induced in the right cheek pouches of 8- to
12-week-old hamsters by a previously described method (17)
using 9,10-dimethyl-l,2-benzanthracene (DMBA, Eastman Or
ganic Chemicals, Rochester, New York). When all treated
pouches had developed tumors, treatment was stopped and,
after a pause of at least one week, tumor measurements were
begun.
A spring caliper was used to measure the longest tumor
CANCER RESEARCH VOL. 28
 Cell Proliferation and Carcinogenesis

diameter and two diameters at right angles to it. Volumes RESULTS

were calculated assuming either spherical or ovoid shapes. When
Latent Period. Repeated applications of DMBA produce a
the average tumor volume had doubled several times, the ham
sters were injected i.p. with thymidine-3H (approx. 0.75 series of clinically observable changes in the mucosal surface
/iC/gm, sp. act. 0.36 c/mmole, The Radiochemical Centre, of the cheek pouch (18). The first reaction was a severe
Amersham, Bucks, England) and killed at intervals thereafter. purulent inflammation which resolved regardless of the con
Autoradiographs were prepared from freshly fixed tissue by tinued presence of carcinogen. Restitution of morphologic in
a modification of the dipping technic. Exposures were deter tegrity was followed by a thickening of the epidermis, as
evidenced by increasing opacity. Although these changes
mined by the labeling density of test autoradiographs developed
occurred in all experimental animals prior to tumor develop
two weeks after dipping. Ideal exposures were planned to yield
ment, the time scale of premalignant changes exhibited a large
an average of 15 grains per labeled nucleus.
degree of variation.
In one experiment, autoradiographs were prepared from
Visible tumors developed first in the DEA hamsters and the
cheek pouches of 7 littermates between 6 and 12 hours old.
These animals were killed at intervals over a 10 hour period.
The percent of cells and mitoses labeled at different times TUMOR LATENT PERIODS
after a pulse of thymidine-3H was determined by counting a
MEAN 10.75
minimum of 3000 cells and 100 mitoses/sample. In most cases CREAM
the sample size was at least doubled. Although one investigator
9
scored the autoradiographs, repeated counts of a single slide
or counts of different sections taken from a single sample were
consistent.
7
Mean transit times through the cell cycle were determined
from plots of labeled mitoses/mitoses (LM/M) and labeled 5
cells/cells (LC/C) at various times after the injection of thy-
oc.
midine-3H. In normal cheek pouches these indices were derived a 3
exclusively from the basal cell population, which was believed
to be the important if not the only source of cell production I
during steady state conditions. J l
However, the relative paucity of kinetic information, differ 9 -GOLDEN MEAN 10.0
ences in physical properties and histologie organization made
it impossible to identify comparable cell populations in neonatal co 7
pouches or in tumors. In these tissues it was necessary to score LU
autoradiographs without reference to a defined proliferative I
O 5
population. Premalignant hyperplastic epithelium occupied the o
spaces between tumors in treated pouches and was also scored o
in its entirety for similar reasons.
CL
3
U-
Ts, the mean DNA synthesis time, was estimated from the
o I
area under the first wave of labeled mitoses (5, 23). Tc, the
mean cycle time, was measured directly as the difference be
ce. l I l
LU
tween the 50% intercepts of the ascending limbs of successive CD 9 -DEA MEAN 7.3
waves of labeled mitoses in tumors. Mean transit time through
the cell cycle in tumors was also calculated from the measured 7
values for Ts and LC/C, assuming that all tumor cells were
contributing equally to exponential growth of the tumor mass.
5
As previously reported (23), growth under these circumstances
can be described by the following quadratic equation:

T, + ^ T* + 2(2T„2mri+ 77)LC/C
T
1 c—
— I
2.88 (LC/C)
0
Although there were morphologic indications that not all tu 2 4 6 8 IO 12 14 16 18
mor cells were proliferating, the discrepancy between measured WEEK OF APPEARANCE
and calculated values of Tc provided additional evidence. (POST TREATMENT)
Tc in cell populations other than tumors was calculated from
the measured values of Ts and LC/C according to an extension Chart 1. Histograms showing time distributions of the appear
of Wright's hypothesis (28), which postulates that the fre
ance of the first tumor per pouch in each of the three strains of
quency of cells in a phase of the cell cycle is proportional to hamsters. The mean for dark-eared partial albinos is significantly
the transit time through that phase. different from the other groups at the 5% confidence level.

MAY 1968 899

Allan B. Reiskin and Roger J. Berry
mean latent period for the group (7.3 ±2.8 weeks) was sig
nificantly different from the mean latent period for cream and
golden strain animals, which were 10.0 ±0.49 and 10.7 ±0.44
weeks respectively. Inspection of Chart 1 suggests that the
DEA latent periods were more evenly distributed than they
were in either of the other strains. The apparent segregation of
latent periods in cream and golden hamsters had no statistical
significance, in the context of the number of experimental an
imals on which the analysis was based.
Tumor Growth Rates. Tumor growth was rapid, and three
growth patterns were observed in all strains: (a) most tumors
grew continuously but with changing rates', (b) a few tumors
grew continuously with constant rates; and (c) a few tu
mors went through periods of regression although growth was
always resumed. When the volumes of tumors from each strain
were pooled and plotted as the log mean of tumor volume at
different times after the beginning of treatment, growth ap
peared to have an exponential mode (Charts 2-4). Regression
lines were fitted to these data by the least squares method, and
from them doubling times of 4.7 ±0.6, 12.3 ± 1.1, and 9.3
±0.5 days were calculated for DEA, golden, and cream tumors
respectively. The difference between DEA hamsters and the
other strains was significant P < 0.05).
Epithelial Proliferation in the Cheek Pouches of Untreated
Neonatal and Adult Hamsters. Since the mean survival time of
the newborn hamsters was reduced dramatically by experi
mental manipulation, it was impossible to measure Tc directly.
However, a value for this parameter was calculated using
Wright's hypothesis (23, 28) and the measured values of T,
and LC/C. T,, measured from the area under the first wave
of labeled mitoses in Chart 5, was 6.7 hours. Although the num
ber of samples was small, they fully define a first wave. The
calculated Tc of 29.2 hours is excessively long, since the de
nominator of the labeled cell/cells fraction unavoidably in
cluded an undefined number of nonproliferating cells.
In the adult hamsters, the proliferating compartment was
better defined, and less error was associated with the calculated
values for Tc of 141 and 155 hours in Golden and DEA an
imals respectively. The mean transit times through DNA syn
thesis (Charts 6, 7) were 9.9 and 9.3 hours, both considerably
longer than in the neonatal hamsters. All of the labeled mitoses
curves in untreated animals were reasonably symmetrical, and
they differed from each other primarily with respect to the
length of the plateaus.
Kinetics of Carcinogen-induced Hyperplasia. The areas of
the treated cheek pouch which surround the tumors are ir
regularly hyperplastic. Earlier experiments have indicated that
this histologically nonmalignant tissue gives rise to additional
new tumors as time passes (19). From autoradiographs of this
material, it was possible to define a wave of labeled mitoses
(Chart 8). The wave was qualitatively similar to those ob
tained from normal basal epithelium of the adults with two
main exceptions. First, the data tended to scatter, and second,
the descending limb of the curve dropped less steeply than it
did in the previous curves. T,, measured from the area under
the wave, was 10.3 hours.
As indicated in Table 1, the percent of cells labeled by a
pulse of thymidine is approximately twice as great as in the
900
untreated cheek pouches, and Tc calculated from the labeling
index and T, equal 61 hours. The reliability of this calculation
and its significance will be discussed later.
Cell Kinetics of Chemically Induced Tumors. Tumor his
tology was similar in all experimental groups. Only squamous
cell carcinomas were observed, and these could easily be sep
arated into four types according to the degree of differentia
tion. The least differentiated and most rarely encountered tu
mor was composed of spindle-shaped cells that could be seen
in transitional stages streaming away from the basement mem
brane. The second type was composed also of primitive epi
thelial cells and had, in addition, significant areas of necrosis.
The two remaining histologie types, which accounted for more
than 90% of the tumors, were differentiated and produced
moderate amounts of keratin. They were distinguished from
each other by the degree of uniformity of the basal cells.
The grain density of tumor cell labeling by tritiated thy
midine varied from tumor to tumor. A single tumor from one
animal displayed no thymidine uptake, although mitotic figures
were apparently normal and control tissues from the animal
showed moderate amounts of label. Each point in Charts 9-11
represents a single tumor. Most of the points at a given time
GOLDEN TUMORS
1000
UJ
O
er
O
100
<t
_l
LU
ec.
IO
50 90 130 170
DAYS FROM THE BEGINNING OF TREATMENT
Chart 2
Charts 2-4. The volume of one tumor in each pouch was meas
ured and the log mean of the combined volumes was plotted
against the time after treatment commenced. DEA, dark-eared
partial albino hamsters.
CANCER RESEARCH VOL. 28

interval are multiple tumors from a single animal but almost
every time interval includes tumors from a second and, oc
casionally, a third animal as well. Although there was not
enough material for a rigorous analysis, the variation between
tumors from different animals did not seem to exceed the
variation among tumors in the same animal. Tt was 6.4, 7.4,
and 7.9 hours in the DEA, cream, and golden animals respec
tively. The Tc measured directly from the labeled mitoses curves
was 11 hours for DEA tumors and 16 hours in both creams
and goldens.
DISCUSSION
In other studies, dealing primarily with carcinogenesis,
attempts have been made to correlate kinetic parameters with
oncogenic sensitivity. Some investigators (9) have reported
an enhanced response to chemical carcinogens when cells were
in the DNA synthetic phase of the cell cycle at the time of
treatment. Others have indicated that DNA synthesis may be
a required step in the carcinogenic process (1, 9). Although
these experiments were not designed to do so, one might have
expected that the interstrain comparisons in this report would
corroborate earlier experiments. However, the subtlety of ki
netic differences between strains makes it impossible to draw
firm conclusions about the effect of these differences on sen
sitivity to chemical agents. It is interesting to note that Morris
(17) reported a reduced latent period in younger animals, and
this report, as well as preliminary data on weanling hamsters,
CREAM TUMORS
1 1 1
1000
111
ce
o
00
LU
ce
IO
i
50 90 130 170
DAYS FROM THE BEGINNING OF TREATMENT
MAY 1968
Cell Proliferation and Carcinogenesis
indicates that, during normal growth, a higher proportion of
proliferative cells are in DNA synthesis.
The mean transit time through DNA synthesis was shorter
in neonatal pouch epithelium (6.7 hours) and in epithelial tu
mors (6.4-7.9 hours) than in the normal pouch epithelium of
adults (9.9-10.3 hours). Preliminary experiments have indi
cated that, in adult animals, DNA synthesis may also be
shortened during regeneration, and, therefore, T, is not an ef
fective indicator of malignancy as was previously suggested
(23). In association with these reductions in synthesis time,
there were concomitant reductions in other phases of the cell
cycle, which had the net effect of increasing the relative size
of the population in synthesis.
The accuracy of the calculated kinetic parameters depends
primarily on two factors, the adequacy with which the equa
tions describe the kinetic phenomena and the reliability of the
data used. Three measurements (Tt, LC/C, and M/LC) have
been used in the calculations described in this report and each
requires some clarification. T, was measured directly by the
labeled mitosis method in each experimental cell population.
Although it is difficult to apply meaningful confidence limits
to the data, there are clear, resolvable differences between
neonatal and neoplastic epithelium on the one hand and pre-
neoplastic and undisturbed adult epithelium on the other. The
repeatability of scoring was tested, but of equal importance
was the assumption made in using the equations that all cells
DEA TUMORS
100
ce
o
i ,0
ce
30 50 70 90
DAYS FROM THE BEGINNING OF TREATMENT
901
Allan B. Reiskin and Roger J. Berry

NEWBORN DEA EPITHELIUM were actively dividing. The correctness of the assumption is
clear only in the case of LM/M, since there is visible proof
100
of proliferation. The LC portion of the LC/C and M/LC
fraction may be misleading if synthesis occurs without cell
division in significant numbers of cells.
i: 75
In scoring autoradiographs of neonatal epithelium, it was
impossible to avoid the inclusion of some differentiated and,
50
therefore, nongrowing cells. If a correction factor could be
2 measured and applied, it would reduce the calculated Tc. In
O untreated adults, information obtained from chronically infused
animals suggested that only and all basal cells proliferate.
Tc was measured directly in the tumor cell populations and
could be used together with the measured values of T, to cal
culate a more reliable LC/C index. In the epithelial hyper-
O 5 IO 15 20 25 30 35 plasia induced by the carcinogen, it was not possible to dis
HOURS AFTER INJECTION OF TRITIATED THYMIDINE tinguish between dividing and differentiated or other nonpro-
liferating cells, except by the criteria of thymidine uptake and
Chart 5. Labeled mitoses curve of cheek pouch epithelium from visible mitosis. It was clear that LC/C was not an accurate
newborn dark-eared partial albino (DEA) hamsters. index of proliferation, and, in contrast to other situations, it
was impossible to predict the direction or the extent of error.
DEA NORMAL BASAL EPITHELIUM
The calculated cell cycle in this case must be confirmed by
100
direct measurement before it can have significance. If the cal
culated value is grossly incorrect, and the duration of the cell
cycle as well as of DNA synthesis are similar to normal values,
then the increases in labeling may indicate a failure or sup
pression of differentiation. Such a finding would provide con
vincing support for the concept of delayed maturation as a
part of the carcinogenic mechanism (2).
It should be noted that small differences in Ts (0.6 hours)
and LC/C (1.0%) result in apparently large differences in the
calculated cell cycles. These differences are not likely to be
real but, as long as the data are interpreted with regard to the
input parameters, valid distinctions and comparisons can be
5 10 15 20 25 ÕO made.
HOURS AFTER INJECTION OF TRITI ATEO THYMIOINE
The present results clearly indicate that, in similarly treated
hamsters, significant differences occur with respect to tumor
Charte latent period and growth rate as a function of strain. This
finding was anticipated, since the strain dependence of latent
Charts 6, 7. Labeled mitoses curves of epithelium from cheek periods and tumor growth rates has been demonstrated in other
pouches of adult dark-eared partial albinos (DEA) and Golden
hamsters. DEA PRENEOPLASTIC EPITHELIUM

100
GOLDEN NORMAL BASAL EPITHELIUM
100

0 5 10 15 20 25 30
HOURS AFTER INJECTION OF TRITIATEO THYMIDINE
5 10 15 20 25 30 35
HOURS AFTER INJECTION OF TRITIATEO THYMIDINE
Chart 8. The first wave of labeled mitoses occurring in the cheek
pouch epithelium of adult dark-eared partial albino hamsters
Chart 7 (DEA) during carcinogen-induced hyperplasia.

902 CANCER RESEARCH VOL. 28


Table 1
(hours)TissueDEA Time periods
basaiepitheliumGolden
basalepitheliumDEA
newbornepitheliumDEA
hyperplasticepitheliumDEA
tumorGolden
tumorCream
tumorT,9.39.96.710386.487.97.4T»i142.4126.420.647.411.010.9172T»21.142.61.562.031.971.761.74rm2.151.60.271.180.560.600.66Tc
Autoradiographic indices determined for normal, hyperplastic, and tumor cells. DEA, dark-
eared partial albino hamsters.
Tm _ Mitoses
~f7 ~ Labeled cells
"T, Labeled cells
= LC/C
T, CUE
VT i
—
2.88(LC/C)
mammalian species (7). A comparison of cell transit times,
latent periods, tumor growth rates, and 'crude estimates of
growth fractions (15) are presented in Table 2.
In spite of the fact that the cheek pouch was selected as the
experimental site because it seemed less susceptible to physio
logic and functional variation, kinetic changes resulting from
an altered local environment after carcinogen treatment cannot
be ruled out. For this reason, and because the tumors were
differentiated (known to have a growth fraction), it was pos
sible to anticipate a complex relationship between tumor growth
and tumor cell cycles (16).
Tumor growth rate at any given time is related to the growth
fraction and to cell death or loss, as well as to generation time,
and it is possible to make rough estimates of the relative sig
nificance of these factors. The labeled mitoses method for
measuring generation time parameters requires some rate
coherence of the labeled cells as they progress through the
cycle. The shape and sharpness of the waves reflects the
amount of rate variation in the cohort (21). Although the tu
mor-labeled mitoses curves did show some scatter, the waves
are sufficiently distinct and symmetrical to indicate that rate
differences within and between tumors occurs only within
limits. It is reasonable to conclude that a major source of varia
tion in growth rate is related to cell loss or the growth frac
tion.
In its simplest form, the growth fraction concept implies one
way movement of cells from a growing to a nongrowing form.
There are, however, several precedents for two-directional
movement of cells between growing and nongrowing compart
ments in both normal and malignant tissues (Griswold et al.,
personal communication ; L. J. Talmach, L. A. Palmer, and W.
E. Powers, personal communication; 10-12, 13). In the cheek
MAY 1968
Cell Proliferation and Carcinogenesis
labeled
calcu T, cells labeled
measured
(LC/C)6722.915.711.0 cell020.170.040.110.060.050.06
lated155.0»141.8«295«61.1«21.9621.1"27.8»%
24.916.0
31.016.0
23.0mitosesi.ii.i131.81.61.61.5Mitoses/
pouch tumors, two-directional movement is not clearly estab
lished, but the abrupt changes in growth rate (including re
gression) and the presence of labeled and mitotic cells in other
wise differentiated areas strongly suggests that it does occur.
The mechanism which regulates the participation of prolifera-
tively capable cells in the division cycle is poorly understood.
In normal tissues the phase at which cells enter or leave the
cycle varies and may be a function of tissue specific factors.
Liver cells, for example, leave the cycle in the early postmitotic
period and enter the G0 state (13). DNA synthesis and other
biochemical events must occur before these cells can divide if
stimulated. In contrast, some epidermal cells leave the cycle
in the Go phase and are ready for division with little metabolic
preparation (11). Since it is not clear which, if either, situation
applies to tumor cells, it is advantageous to avoid using the G0
term which implies a specific biochemical pathway and to sub
stitute a more general term, T0, to describe any proliferatively
capable nondividing cell.
The existence of a T0 compartment has important implica
tions for radiotherapeutic or chemotherapeutic management of
neoplastic disease as well as for tumor growth analysis. If
changes of fluctuations in growth rate are primarily related to
changes in the relative size of the T0 compartment, rather than
to changes in the duration of the cell cycle, then the task of
obtaining kinetic information on human tumors with conven
tional technic may not be as formidable as it has seemed in the
past. On the other hand, the possible existence of T0, or out-
of-cycle and, perhaps, resistant tumor cells, represents an un
known and potentially hazardous quantity. Finally, the T0
state may invalidate growth curve extrapolations and require
new approaches to tumor growth analysis.
903
Allan B. Reiskin and Roger J. Berry
DEATUMORS
Table 2
100

Hamster
strain period
tissuesDEACreamGoldenroofuntreated(wk.)7.310.010.7DT
cells155.0141Latent cells11
(days)4.79.3123r.dtumor
H16
H16
HGF0.070.050.04
Interstrain comparison of cell cycles, latent periods, tumor dou
bling times (DT), and tumor growth fractions (GF). Growth
fractions were calculated from the relationship between Tc and
/ln(l + GF) In2\
the doubling time >AfI To ~ DT/ I See Table 1 for abbre-

viations.
5 10 15 20 25 30 35
HOURS AFTER INJECTION OF TRITIÄTEDTHYMIDINE
ACKNOWLEDGMENTS
Chart 9
The authors wish to thank S. A. Tyler and M. H. Dipert for
Charts 9-11. Labeled mitoses curves for chemically induced their collaboration in the statistical evaluation of the data and,
tumors in the cheek pouches of adult dark-eared partial albino in addition, all those who contributed editorial comment and
(DEA), Golden, and Cream hamsters. Each point represents a assistance.
single tumor.

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GOLDEN TUMORS
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