[CANCER RESEARCH 46, 2344-2348, May 1986]
Role of Spontaneous Transformation in Carcinogenesis: Development of
Preneoplastic Rat TrachéalEpithelial Cells at a Constant Rate
David G. Thomassen
Laboratory of Experimental Pathology, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701
ABSTRACT
The rate of spontaneous transformation of normal rat trachéalepithe
lial (RTE) cells to preneoplastic enhanced growth (EG) variants was
estimated in serum-free culture. Spontaneous transformation of RTE
cells has previously been observed, but an accurate estimation of the rate
of change has not been possible due to the use of serum and feeder cells
in the cultures which prevents both unlimited RTE cell proliferation and
an accurate determination of the number of cells at risk. RTE cells were
plated in serum-free medium and were switched to serum-containing
medium at various times during the first 23 days of culture. In serumcontaining medium, normal RTE cells cease proliferation, while EG
variants continue to proliferate. The fraction of RTE cell colonies which
developed into EG variants increased with time to a maximum of 15%
when selection was imposed 5 to 23 days after plating. The number of
cells per culture also increased during the same time, suggesting a role
for cell proliferation in the spontaneous generation of EG variants. In
contrast to the time-dependent increases in cell number and the frequency
of EG variants, the rate of development of spontaneous EG variants
remained constant with time and was estimated to be 7.5 ±4.1 x 10 "
variants/cell generation. The rate of spontaneous preneoplastic transfor
mation of normal epithelial cells reported here, the rates of spontaneous
progression of preneoplastic and neoplastic cells reported elsewhere, and
the association between cell proliferation in vivo and increased cancer
risk are consistent with the hypothesis that spontaneous changes play a
role in the multistep progression of cells to cancer.
INTRODUCTION
Neoplastic transformation in vivo and in vitro is a progressive,
multistep process (1-3), but definitive information on the na
ture, origin, and number of the required specific cellular changes
is lacking. The role of carcinogenic agents in neoplastic trans
formation has been studied extensively, and many changes
which occur during preneoplastic progression can be attributed
to interactions of these agents with cells in vivo or in vitro.
However, changes can occur during the progression of cells to
neoplasia which cannot be attributed to carcinogen exposure.
These changes have been described as spontaneous (4). Spon
taneous changes have most frequently been described in cell
culture, where the prolonged passage of cultured normal rodent
cells (4) usually results in the emergence of neoplastic cells. In
contrast, normal human cells cultured under similar conditions
rarely (5), if ever (6), undergo spontaneous neoplastic transfor
mation. In some cases, changes initially described in cell culture
as spontaneous have been attributed to specifically identifiable
environmental factors (4). However, the development of spon
taneous changes /'// vivo and in vitro can be affected by the
fidelity of DNA replication and the maintenance of normal
DNA structure and activity. The roles of DNA repair, replica
tion, and recombination in spontaneous mutagenesis have been
well documented (7). Similarly, their roles in neoplastic trans
formation should be considered. An understanding of the origin
of spontaneous changes and their role in progression to neopla
sia is important in the interpretation of models and mechanisms
Received 8/9/85; revised 1/13/86; accepted 1/30/86.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
of carcinogenesis.
Quantitative studies on the rates of spontaneous transition
between normal and preneoplastic cells or between preneoplas
tic and neoplastic or malignant cells can provide basic infor
mation needed to determine the role(s) of spontaneous changes
in the multistep process of carcinogenesis. Such rates have been
estimated in vivo (8) from the age-specific tumor incidence data
of individuals who have a dominantly inherited cancer syn
drome such as retinoblastoma or polyposis coli. These estimates
are expressed as rates of tumor development per year or per
(human) generation but not per cell at risk, since the number
of cells at risk in vivo cannot be accurately estimated. However,
many in vitro cell transformation systems permit determination
of the number of cells at risk. Thus, rates of change per cell at
risk can be determined. Determinations have previously been
made using preneoplastic or neoplastic cell lines in culture for
the rates of spontaneous change from anchorage dependence to
independence (reviewed in Ref. 9) from nontumorigenic to
tumorigenic (10), and from nonmetastatic to metastatic (11).
In this paper, the rate of spontaneous preneoplastic transfor
mation is determined for primary RTE' cells in serum-free
culture, using the Luria-Delbriick fluctuation analysis (12). As
previously described (13), treatment of RTE cells with carcin
ogens in vivo (14) or in vitro (13, 15) induces heritably altered,
preneoplastic cells, termed EG variants, which have the capacity
to proliferate /// vitro under conditions where normal RTE cells
fail to do so. These variants are nontumorigenic but can become
tumorigenic with additional time in culture (13, IS). Sponta
neous transformation of RTE cells to EG variants was observed,
and it was proposed that the frequency of spontaneous EG
variants was a function of cell proliferation (13). However, an
accurate estimation of the rate of this spontaneous transfor
mation was not possible because of the use of serum and the
presence of feeder cells. In the present study, a serum-free and
feeder cell-free culture system for the clonal proliferation of
normal RTE cells was developed to eliminate the inhibitory
effects of serum in long-term cultures and to eliminate the use
of feeder ceils (13). Selection for preneoplastic EG variants was
imposed by switching cultures from serum-free to serum-con
taining medium to mimic previously used selective conditions
which consisted of selective removal of feeder cells from the
cultures and the same serum-containing medium used here (13).
Using these conditions, the rate of spontaneous transformation
to EG variants can be estimated. The hypothesis that sponta
neous changes can play a role in the multistep progression of
cells to cancer is examined with respect to this rate of sponta
neous preneoplastic transformation, rates of spontaneous pro
gression of preneoplastic and neoplastic cells, and the associa
tion between cell proliferation in vivo and increased cancer risk.
MATERIALS
AND METHODS
Cells and Cell Culture. RTE cells were obtained from 7- to 8-wk-old
male Fischer 344/NCR rats (specific pathogen free; Animal Produc1The abbreviations used are: RTE, rat trachea! epithelial; EG, enhanced
growth.
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SPONTANEOUS
TRANSFORMATION
tion, Frederick Cancer Research Facility, Frederick, MD) as previously
described (13). Primary RTE cells were cultured in serum-free medium
composed of Ham's F-12 medium (GIBCO) modified by increasing the
Ca2+ to 0.8 HIM and adding 15 HIM4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer (Utrol; Calbiochem-Behring) and supple
mented with 1% (v/v) bovine pituitary extract (16), bovine serum
albumin (500 /ig/ml) (essentially globulin free; Sigma), 10"' M cholera
toxin (Sigma), epidermal growth factor (5 ng/ml) (Collaborative Re
search, Waltham, MA), 5 x 10~5 M ethuno lam ine (Sigma), hydrocor
tisone (0.1 Mg/ml), insulin (5 /ig/ml) (Calbiochem, La Jolla, CA), 5 x
l O"5M phosphoethanolamine (Sigma), transferrin (5 /¿g/ml)(Sigma),
penicillin (100 units/ml), and streptomycin (100 tig/ml) (GIBCO).
Stock solutions were prepared as described (16).
Medium for selection of preneoplastic EG variants consisted of
Ham's F-12 medium supplemented with 5% fetal bovine serum (Lot
100104; Advanced Biotechnologies, Inc., Silver Spring, MD), insulin
(1.0 Mg/ml), hydrocortisone (0.1 Mg/ml), and antibiotics (13). All cul
tures were fed twice weekly with the appropriate medium.
Transformation Assays. The development of EG variants with time
in culture was examined by plating RTE cells in serum-free medium
and then selecting for EG variants by switching, at various times after
plating, to serum-containing medium which will not support the con
tinued proliferation of normal RTE cells. RTE cells were plated in 60mm-diameter tissue culture dishes (Lux, Naperville, IL) at 6000 (Ex
periment 1) or 2500 (Experiments 2 and 3) cells per dish. At the
specified times after plating in serum-free medium, 5 to 20 dishes of
RTE cells were switched to serum-containing medium for an additional
4 wk to select for EG variants. The number of cells per culture continued
to increase for several days after switching from serum-free to serumcontaining medium, increasing the number of cells at risk for sponta
neous change. Estimates of the rate of spontaneous transformation to
EG variants were, therefore, based on the maximum number of cells at
risk in each set of cultures. A determination of the number of cells per
culture was made on representative cultures on the day of switch to
serum-containing medium and for up to 7 days following each switch,
by which time the number of cells per culture was stabilized or decreas
ing. The colony-forming efficiency of RTE cells in serum-free medium
was determined as described (13) 10 days after the RTE cells were
plated. The frequency of spontaneous EG variants was determined for
each set of cultures switched to serum-containing selective medium at
different times after plating. The frequency was calculated as the
fraction of RTE cell colonies which formed in serum-free medium by
Day 10 of culture which became EG variant colonies 4 wk after being
switched to selective medium.
Determination of the Rate of Spontaneous Transformation. The rate
of spontaneous transformation was estimated for each time point using
the equation from Luria and Delbrück(12).
r = a/V,ln (CaNJ
where r is EG variants per culture, a is rate of spontaneous transfor
mation of RTE cells to EG variants (variants/cell generation), N, is
maximum number of cells per culture, and C is number of cultures.
Since r, N„
and C are known values, the equation is solved for a using
the method of Capizzi and Jameson (17) which has a computergenerated table for calculation of a. The rate of spontaneous transfor
mation is expressed as EG variants/cell generation, where one cell
generation is defined as the production of two cells from a single cell
by mitosis. Although all cell lineages do not contribute equally to the
total number of cells in a population due to unequal rates of cell
division, the total number of cell generations (mitoses) which have
given rise to a cell population is theoretically equal to the total number
of cells at the time of testing minus the initial number of colonyforming cells.
OF RTE CELLS
subsequent
0 to 7 days (average,
3.6 ±2.6 days), after which
the RTE cells flatten and begin to slough from the dishes. The
numbers of RTE cells in cultures on each day of switch to
serum-containing medium are shown in Table 1, and the max
imum numbers of cells in these same cultures are shown in Fig.
IA and Table 1. The maximum number of cells per culture
increased exponentially during the first 10 to 11 days after
plating with a population doubling time of approximately 20 h,
after which a plateauing was seen on Days 11 to 23.
After being switched to serum-containing medium at speci
fied times, cultures were maintained for an additional 4 wks
and the fraction of RTE cell colonies becoming EG variant
colonies was determined (Table 1, Fig. \B). The fraction of
colonies becoming EG variants steadily increased from Days 5
to 11, doubling approximately every 1.5 days. From Days 13
to 23, the fraction of colonies becoming EG variants increased
much more slowly, doubling only about once a week.
The rate of spontaneous transformation of RTE cells to EG
variants was estimated for each day of switch to serum-contain
ing medium (Table 1; Fig. 10 and was based on the maximum
number of cells present in the cultures after switching to serumcontaining medium (Table 1; Fig. IA). To determine if varia
tions in the rate were time dependent, the hypothesis that the
rate remained constant with time was tested by regression
analysis. The null hypothesis that the slope of the regression
line of rate versus time was significantly different than zero was
tested for rates calculated from Days 5 to 23. A probability of
P < 0.05 would result in rejection of the null hypothesis. The
slope of the regression line did not differ significantly from zero
for Days 5 to 23 (P = 0.39), suggesting that the rate of
spontaneous transformation of RTE cells to EG variants re
mained constant with time. The average rate of change for Days
5 to 23 was 7.5 ±4.1 X 10~6EG variants/cell generation. Rates
could not be accurately estimated at earlier times due to large
errors in determining cell numbers at early times and due to
the small number of variants obtained.
DISCUSSION
The development of spontaneous preneoplastic EG variants
from normal RTE cells in culture has been analyzed to gain
insight into the role of spontaneous transformation events in
carcinogenesis. The occurrence of spontaneous changes in cells
is theoretically dependent on cell proliferation for the genera
tion and amplification of stable genetic variants. In the devel
opment of spontaneous EG variants described here, the fraction
of colonies becoming EG variants increased with time when
selection was imposed 5 to 23 days after plating (Table 1; Fig.
IB). The number of cells per culture also increased during the
same period of time (Table 1; Fig. ÃŒA),
suggesting a role for
cell proliferation in the spontaneous generation of EG variants.
In contrast to the time-dependent increases in cell number and
the frequency of EG variant colonies, the rate of development
of spontaneous EG variants remained constant with time (Fig.
1C; Table 1) and was estimated to be 7.5 ±4.1 x 10~6variants/
RESULTS
cell generation. New EG variant cells arose at a constant rate
within expanding colonies of normal RTE cells, resulting in the
increased frequency of colonies scored as EG variants with
time. It is important to note the high frequency of EG variant
colonies (>10%) which resulted from a relatively low rate
(<10~5/cell generation) of spontaneous change. The combined
When selection for EG variants is imposed (13) by switching
cultures from serum-free to serum-containing medium, the
number of cells per culture increases 1- to 14-fold during the
effects of cell proliferation, cell accumulation, and a constant
rate of spontaneous change at the cellular level yielded high
frequencies of variant colonies at the population level, which
2345
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SPONTANEOUS
Fig. 1. Growth and spontaneous preneoplastic transformation of RTE cells in serumfree culture. A, maximum number of RTE cells
per culture: B, the fraction of RTE cell colonies
which become EG variants; C the rate of
spontaneous development of EG variants, cal
culated as function of the time when cultures
were switched from serum-free to serum-con
taining selective medium. Experiment l (D), 2
(O). 3 (A).
TRANSFORMATION
OF RTE CELLS
! ¡10-
10.0-
105
C
g*
n
if
f-?
Ä O
n O
Il
°
1.0
&•£
¡I
103
10
15
20
25
i
0.1
0
105
10
15
20
25
05
Days in Serum-Free Culture Before Switch to Selective Medium
Table I EC variants and estimated rates of their spontaneous development in cultures of RTE cells as a function of time of switch to serum-containing selective
medium
Primary RTE cells were plated in serum-free medium. At various times after plating, sets of cultures were switched to 5% serum-containing medium and were
scored for EG variants 4 wk later. The number of colony-forming cells per culture was determined 10 days after plating. The frequency of spontaneous EG variants
was determined by dividing the number of EG variants found per culture at the end of the experiment by the number of colonies found per culture on Day 10. The
maximum number of cells per culture and the day on which that number of cells was found were also determined for calculation of rates of spontaneous transformation.
The rates of spontaneous transformation of RTE cells to EG variants were estimated for each day of switch to serum-containing medium, using the equation r aN,\n(CaN,). where r is EG variants/culture, a is rate of spontaneous transformation of RTE cells to EG variants (variants/cell generation), N, is maximum number
of cells per culture, and C is number of cultures.
ofcells/cultureon
ofExperiment*
switch1
Day
ofcells/culture(x
coloniesbecomingEG ofspontaneoustransformation(x
dayof
IO"5)(day
switch(x
variants(%)<0.151.41.83.65.80.41.21.63.31.41.910.711.315.0E
IO'5)0.170.761.431.792.860.020.262.202.950.030.060.260.410.63Maximumno.
of
maximum)*0.74(10)2.2(14)2.15(16)2.6(19)2.9
IO6)4.96.17.99.96.65.75.86.420.08.7
7101316232
579113
(23)0.29
(24)7.6
(38)0.62
(9)1.6(10)2.2
(5)1.89(17)2.5(15)5.12(41)0.35
(9)3.0(12)0.11
57131619No.
(12)0.43(14)1.63(20)3.41 (7)0.74(14)4.1
(78)4.27
(19)4.25
(64)5.75
(22)EGvariants/dish(total)0(0)1.8(9)2.4(12)4.8
(92)Fractionof
" The number of RTE cells plated per culture and the colony-forming efficiencies for Experiments 1 to 3 were, respectively, 6000 and 2.2%, 2500 and 6.2%, and
2500 and 1.5%.
* Maximum number of cells per culture after switch to serum-containing medium and the day on which that number of cells was observed.
can make a significant contribution to transformation in cell
culture and theoretically in vivo.
Estimates of the rate of spontaneous transformation will be
affected by a number of factors. Estimates will be affected by
errors in determing the number of cells at risk per culture. The
maximum number of cells potentially at risk for spontaneous
transformation is equal to the total number of attached cells
plus the number of cells lost from cultures prior to determina
tions of cell number. The numbers of cells lost from cultures
can be estimated from the numbers of floating cells isolated S
to 23 days after plating. Cultures of RTE cells in serum-free
medium lose < 5% of their total attached cells per week (data
not shown) and only begin losing large numbers of cells more
than 1 wk after being switched to serum-containing selective
medium. This correction would result in < 10% increases in
the maximum number of cells at risk in cultures switched after
3 wk in serum-free medium (early losses will contribute little
to corrections at late times, since the largest numbers of cells
are lost at the latest times) and smaller increases in the number
of cells at risk at earlier times. These changes in cell numbers
would decrease estimates of the rate of spontaneous transfor
mation by an equivalent amount (i.e., < 10%). However, these
changes are much less than the overall standard error (55%)
for the rates estimated using only attached cells as the total
number of cells at risk and would, therefore, not significantly
affect the results.
The rates and frequencies reported here may also be slightly
underestimated. Each EG variant colony is assumed to arise
from one spontaneous transformation event within an expand
ing RTE cell colony. However, more than one RTE cell in a
colony could transform into a variant cell, although only one
EG variant colony would develop, resulting in an underestimate
of the rate of spontaneous transformation. Such multiple trans
formation events in single colonies are not likely to occur too
frequently, since > 85% of RTE cell colonies do not become
EG variant colonies and thus have not even had single trans
formation events.
If the development of EG variants depends on RTE cell
proliferation as proposed here, effects of cessation of prolifer
ation should be considered. When normal RTE cell prolifera
tion stops, the number of cells per culture will remain constant
or slowly increase as rare EG variant colonies continue to
expand. Similarly, the fraction of RTE cell colonies which
become EG variant colonies will remain constant after all
variant cells which arose before proliferation stopped have
become EG variant colonies. Finally, calculated rates of spon
taneous transformation will also remain constant when cell
proliferation stops, since the rate is based on the total number
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SPONTANEOUS TRANSFORMATION
of cells and EG variant colonies, both of which will have become
constant. Thus, calculations of rate at late times when there are
no increases in the numbers of RTE cells or EG variants will
be artifactual and will only reflect the steady state which has
developed and not a dynamic state of new variant development.
The experiments reported here represent a dynamic state of EG
variant development, since there are increases in the numbers
of cells at risk [see (number of cells/culture on day of switch)
versus (maximum number of cells/culture), Table 1] and the
fraction of colonies which have become EG variants (Table 1;
Fig. IB) over most of the time period examined.
Although it is difficult to demonstrate unequivocally a role
for spontaneous transformation events in carcinogenesis in vitro
or in vivo, an examination of factors important for the devel
opment of spontaneous changes will be useful in evaluating
such a role. The estimated rates of spontaneous change presum
ably related to neoplastic transformation reported here and
elsewhere (see below) can be used to make first order estimates
of frequencies of cells with these changes in vitro or in vivo. In
addition, the relationship between cell proliferation and carci
nogenesis /// vivo will be examined, because of the relationship
between cell proliferation and spontaneous change /// vitro.
Rates of spontaneous change for other neoplasia-related
changes occurring in vitro have been estimated. Rates of spon
taneous change from anchorage dependence to independence
were 3 x 10~7 to 1 x 10~4/cell generation, depending on the
type of cells (mouse, rat, hamster, diploid or tetraploid) and the
kind of semisolid medium which were used (9). The rate of
spontaneous development of tumorigenic variants from a line
of nontumorigenic, anchorage-independent mouse (CAK) fibroblasts was 10~7 variants/cell generation (10). Finally, the rate
of spontaneous development of metastatic variants from the
tumorigenic, nonmetastatic mouse KHT sarcoma cell line was
10~5/cell generation (11). These rates were calculated by the
method described here, although the assays used to identify
variants were clearly different and are a likely source of varia
bility.
These rates represent probabilities of errors that may occur
as a function of cell division regardless of environment and
could, therefore, be used as estimates of risk for spontaneous
change in vivo. They can be used to predict the number of
spontaneously arising tumors expected in an individual with an
inherited gene for retinoblastoma under the assumption that
one additional change is necessary and sufficient for tumor
development (18). Using the equation m = u x jV/ln2, where m
is number of tumors per individual, u is rate of spontaneous
change [10~7 tumor variants/cell generation (10) or 7.5 x 10~6
EG variants/cell generation found here], and N/\n2 is number
of cell generations at risk in two eyes [A' is 4 x 10* cells at risk
in two eyes (19)], the predicted number of tumors in an individ
ual with heritable retinoblastoma is 0.3 or 17 compared to an
observed number of 3 to 4 tumors per individual (20). Thus, in
the case of heritable retinoblastoma, spontaneous changes oc
curring during normal retinal development and having rates
similar to those found /// vitro for neoplasia-related changes
could account for the observed tumor incidence.
If spontaneous changes related to neoplastic transformation
occur as a function of cell proliferation, then cell proliferation
in vivo may increase neoplastic or malignant cell development.
Table 2 lists examples of cell proliferation in vivo and in vitro
which are associated with an increased likelihood of subsequent
neoplastic development. It should be emphasized, however, that
each of these examples of cell proliferation may not always lead
to cancer. Cancer development is a multistage process, and the
OF RTE CELLS
Table 2 Associations between cell proliferation and carcinogenesis
Examples of cell proliferation in vitro and in vivo which are associated with an
increased likelihood of subsequent neoplastic development
Disorders of cell proliferation with increased cancer risk
Polyposis syndromes (23)
Atrophie gastritis (23)
Inherited pancreatitis (23)
Inherited pulmonary fibrosis of the Hamman-Rich type (23)
Ulcerative colitis of the colon (24)
Precancerous disease of the human cervical epithelium (25)
Response to tissue injury and increased cancer development
Hepatic cirrhosis (23)
Nonspecific colon injury and colon cancer (24)
Initiation of bladder carcinogenesis by freeze ulcération(26)
Hyperplastic response to carcinogen-induced toxicity (27)
Wounding and plant tumors (28)
Foreign body tumorigenesis
Foreign body-associated tumors in humans (29, 30)
Foreign body implants in rodents (29)
Tumorigenicity of preneoplaslic cells on plastic plates (31-33)
Promotion
Hyperplastic response, especially sustained hyperplasia (34-36)
Hyperplastic response and Stage I promotion (37)
Wounding as a promoting agent (36)
Normal cell proliferation during development and hereditary cancer
Retinoblastoma, essential change during retinal development (18, 20)
Wilm's tumor, essential change during kidney development (23)
Cell proliferation in vitro or in vivo and neoplastic transformation
Mammary fat pad transplant of hyperplastic alveolar nodules (38)
Serial passage of normal rodent cells in culture (4)
Neoplastic progression of preneoplastic cells in culture (39)
Increased proliferation of epithelial cells in culture (this paper)
spontaneous occurrence of neoplasia-related changes in a pro
liferating cell population will only lead to cancer if other essen
tial change(s) occur or have occurred in the same cells. Cell
proliferation increases the likelihood that spontaneous changes
will occur and, as shown in Table 2, is often associated with
neoplastic development. The association of cell proliferation
with increased risk for both spontaneous change in vitro and
neoplastic development in vivo suggests that spontaneous
change(s) may play a significant role in carcinogenesis.
The immense complexity of cells and organisms and the
changes or adaptations they can undergo make an absolute
determination of a mechanism or a unique pathway of carci
nogenesis theoretically impossible (21). A mult ¡varialomathe
matical model which deals with some of the complexities of
human carcinogenesis has been proposed (22) and encompasses
roles for both spontaneous and induced events and for growth
and differentiation of cells in vivo. The demonstration here that
normal RTE cells in culture undergo spontaneous preneoplastic
transformation at a constant rate which results in high frequen
cies of altered cells supports a proposed role for spontaneous
transformation events in carcinogenesis. The similarly high
frequency of presumed spontaneous change leading to tumor
development in individuals with hereditary retinoblastoma (23)
further emphasizes the potential importance of spontaneous
changes in carcinogenesis. Finally, the relationship between
increased cell proliferation and increased risk of neoplastic
transformation in vivo and in vitro suggests a role for sponta
neous change(s) in this process. The multistage process of
neoplastic transformation is likely, therefore, to be governed by
a balance between changes induced by exogenous or endogenous
carcinogens, changes which occur spontaneously at a constant
rate in dividing cells, and forces in vivo which act to expand,
eliminate, or modulate neoplastic and preneoplastic cell popu
lations.
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SPONTANEOUS
TRANSFORMATION
ACKNOWLEDGMENTS
In memory of and with special gratitude to Dr. Thomas Gindhart
for his encouragement, support, and enthusiasm. I would like to thank
Dr. Umberto Saffiotti, Dr. M. Edward Kaighn, Dr. Nancy H. Colburn,
and Dr. Bonnie Smith for their suggestions and advice; Michael Smart
for technical assistance; and Beverly Bales for preparation of the
manuscript.
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Role of Spontaneous Transformation in Carcinogenesis:
Development of Preneoplastic Rat Tracheal Epithelial Cells at a
Constant Rate
David G. Thomassen
Cancer Res 1986;46:2344-2348.
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