[CANCER RESEARCH 61, 799 – 807, February 1, 2001]
Review
Selected Cell and Selective Microenvironment in Neoplastic Development
Harry Rubin1
Department of Molecular and Cell Biology and Virus Laboratory Life Sciences Addition, University of California, Berkeley, California 94720-3200
“Unlike the situation in earlier periods, clarity does not reside in
reduction to a single, directly comprehensible model, but the exhaustive overlay of different descriptions that incorporate apparently
contradictory notions.”
Gerald Holton, “The Roots of Complementarity,” Thematic Origins
of Scientific Thought.“
Abstract
Recent analysis of genetic alterations in human cancer points to a major
role for selection in neoplastic development but provides few details about
the dynamics of the process. Many such details, however, have emerged
from quantitative studies of spontaneous transformation among mammalian cells in culture. The chief insight of these studies is that there is a
continuous generation of variants in proliferative potential among growing cells that provides the substratum for progressive development to a
frankly neoplastic state when selective growth conditions are persistently
applied. Much of the selection occurs before the cells are capable of
producing discrete neoplastic foci. The varied observations in cell culture
draw attention to analogous features of carcinogenesis in experimental
animals and the development of human cancer.
Introduction
There is an extensive literature on the role of genetic destabilization
in the development of cancer, and it seems to be widely assumed that
it is the driving force in the process. Recent computer modeling, based
mainly on the large body of genetic studies in colorectal cancer,
argues strongly that selection plays the dominant role in the early
stages of cancer with the notable exception of those familial cancers
in which there is a defect in one allele of a mismatch repair gene (1,
2). Increases in natural mutation rates in sporadic cancers are usually
found during later stages of tumor development (3, 4) and are not
considered important in early stages. However, they may play a role
in the exponential increase of cancer rates in aging people if somatic
mutations increase exponentially with age due to the accumulation of
mutations in mismatch repair genes (5). In either case, there has to be
selective growth of altered cells for tumors to develop, and little is
known about the conditions that underlie that selective growth in vivo
or the cellular diversity on which it operates.
By contrast, a considerable body of information has accumulated on
the role of selection in progressive neoplastic transformation in cell
culture including the frequency of selectable variants, the culture
conditions that drive their selection, and the progressive changes in
phenotype that are associated with transformation. The information is
complex and has developed incrementally for over a decade. This
review aims to put the pieces together in a comprehensive picture and
relate it to carcinogenesis in animals and man with particular attention
focused on the role of selection in the process.
Received 7/21/00; accepted 11/21/00.
1
To whom requests for reprints should be addressed. Phone: (510) 642-6617; Fax:
(510) 643-6791; E-mail: [email protected].
Selection in Transformation of Primary Cultures
Most of the knowledge about selection in neoplastic transformation
in culture comes from the process known as spontaneous transformation. It was first discovered in experiments on chemical carcinogenesis of cells from mouse and rat embryos, in which it was found that
the untreated controls eventually produced tumors on inoculation in
syngeneic animals if the cultures had been of sufficient duration (6, 7).
It was later found that increases in the saturation density of mouse
embryo cells occur more rapidly when cells are passaged at higher
population densities (8). Increase in saturation density is associated
with capacity to produce tumors in syngeneic mice: both changes
occurred in BALB/c mouse embryo cells passaged at high density but
not in those passaged at low density (9). In another study using clonal
cultures from hybrid mouse embryos, tumorigenic capacity developed
from both high density passages and LDPs2 but did so a few months
earlier in the former (10). Therefore, in both cases, neoplastic transformation was favored in cultures passaged at high density, which
suggests selection of spontaneously occurring variants with neoplastic
potential that is associated with increased capacity to escape contact
inhibition. It cannot be ruled out, however, that the earlier appearance
of transformed cells in high density passages resulted from the larger
number of cells continually present in the high density cultures as
compared with the low density cultures, which would increase the
probability of detecting neoplastic variants in the former.
Selection in Transformation of Established Cell Lines
Fibroblasts. A permanent line of cells was established by LDPs of
fibroblasts from BALB/c mouse embryos (11). These cells, which
were designated the Balb/3T3 line, maintained a normal sensitivity to
contact inhibition of multiplication at high population densities. They
rarely underwent neoplastic transformation when passaged at low
density (12) but did so more often when suspended in soft agar (13).
The transformed cells had a 10-fold increase in saturation density and
were tumorigenic (14). The increased frequency of transformation in
suspension that inhibits the growth of normal cells suggested a role for
selection in the transformation. However, transformation was still too
infrequent to draw any firm conclusion about the role of selection in
the process.
The permanent line of nontransformed NIH 3T3 cells was established by LDPs of fibroblasts from embryos of a partially inbred strain
of mice (15). These cells were widely used as targets for transformation by putative oncogenes, but they developed transformed foci
spontaneously if left at confluence for extended periods of time (16).
Cells from large, dense foci were highly tumorigenic in nude mice
(17). The spontaneous transformation of the NIH 3T3 cells occurred
regularly at confluence, but this response to growth constraint decreased gradually in repetitive testing of cells that were kept at a
maximal growth rate by frequent passage at low density in high (10%)
concentrations of calf serum (18). After many such LDPs, several
rounds of prolonged constraint at confluence were required before
2
The abbreviations used are: LDP, low density passage; MNNG, N⬘-nitro-Nnitrosoguanidine.
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transformed foci developed (19, 20). The reason for the reduction in
susceptibility to transformation that resulted from LDP may be related
to the observation that transformed cells usually multiply more slowly
at low population density than nontransformed cells (21–23), with the
possibility that cells more susceptible to transformation also exhibit
this tendency and are selected against under maximal growth conditions at low density. If the LDPs were made in 10% fetal bovine
serum, which reduced the growth rate of the cells to about 80% of that
in calf serum, there was a marked increase in transformation (18).
This indicates that selection for transformation and transformability is
effective with even small differences in selective advantage when
maintained over an extended period.
The role of selection in spontaneous transformation is also illustrated by LDPs when the concentration of calf serum is reduced from
10% to 2% (Fig. 1). There is a steady increase in saturation density of
susceptible populations that is detectable when tested after each of the
frequent LDPs in 2% calf serum over a 2-week period (24). No sign
of an increase is seen in parallel LDPs of the cells in the standard 10%
calf serum. To determine whether reducing serum concentration and
maintenance at high population density have an additive effect in
transformation, many cultures were allowed to grow to confluence in
2% calf serum without passage. At intervals of 2 or 3 days corresponding to the frequent LDPs, some of the cultures were transferred
to determine their saturation density. The saturation density on transfer was found to increase steadily for 1 week while the number of cells
in the source culture was still increasing. However, the saturation
density on transfer did not increase further in the following week
when growth of the source culture had markedly decreased (24). This
showed that the somewhat reduced but still exponential growth in
LDPs with 2% calf serum is far more effective in driving transformation than is the stationary state produced at confluence in the same
concentration of serum. It indicates that progressive transformation
requires multiplication, albeit under conditions of constraint that permit selection of those cells that have a growth advantage over the
majority population. However, a small minority of clonal populations
may continue to multiply when the population as a whole reaches its
saturation density; these selected cells would continue to accumulate
Fig. 2. Saturation densities of cells that have been through a 1° assay for 2 weeks in
either 2, 5, or 10% calf serum and then assayed serially in 2% calf serum for the 2°, 3°,
and 4° assays. 1° assays in 2% calf serum, f; 5% calf serum, F; and 10% calf serum, Œ.
The open symbols (E and ‚) represent cultures that had been in 5% or 10% calf serum
in the 1° assay but produced many large dense foci in the 3° and 4° assays, thereby
disproportionately increasing saturation density.
mutations that would drive further selection, culminating in the formation of distinctive transformed foci, each derived from a single cell.
The role of multiplication under constrained conditions in driving
progressive transformation is illustrated by an experiment in which
cells were grown to confluence in 2%, 5%, and 10% calf serum in a
primary (1°) assay (20). The saturation density and the total number
of cell divisions were proportional to the serum concentration. The
cells from the 1° assays in each serum concentration were then serially
assayed three times (2°, 3°, and 4° assays) in 2% serum. Although
they were all in the same 2% serum concentration beyond the 1°
assay, the saturation densities remained progressively higher as a
function of their original serum concentration (Fig. 2). In addition,
large, dense foci appeared in the later assays in proportion to the
saturation density and therefore to the total number of divisions in the
1° assay (Fig. 3). Not only did this experiment confirm the transforming role of multiplication under constraint, but it also showed that
Fig. 1. Changes in saturation density of cells
passaged frequently in high or low concentrations
of calf serum or maintained without passage in a
low concentration of calf serum. NIH 3T3 cells that
had been maintained at their maximal growth rate
by LDPs every 2–3 days were seeded at 105 cells/
dish into each of many 21-cm2 culture dishes in
medium with 10% (A) or 2% (B and C) calf serum
and treated as follows. A, cells were passaged at
2–3-day intervals in 10% calf serum. At each passage, 105 cells were seeded into 20 dishes in 2%
calf serum for growth curves and saturation densities. B, cells were passaged at 2–3-day intervals in
2% calf serum with seeding of 105 cells as described in A in 2% calf serum for growth curves
and saturation densities. C, cells were maintained
without passage in 2% calf serum. On the same
days that cells in A and B were passaged, some of
the cultures in C were trypsinized to set up growth
curves in 2% calf serum. The day on which the
cells were transferred to set up growth curves is
shown next to the appropriate symbol in B and C.
The corresponding curves in A are effectively indistinguishable from one another, with the cells
having undergone no change on frequent passage in
10% calf serum.
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Fig. 3. Photograph of the rate of development and size of foci in four lineages, each
started in 2% or 10% calf serum in a 1° assay as indicated. The photographs of the 2°, 3°,
and 4° serial assays in 2% calf serum are shown.
selection operates on cells before they produce clearly visible, discrete
foci, thus recalling the incipient state of neoplasia described by Foulds
(25, 26).
The power of selection is enhanced by stepwise reductions in calf
serum concentration from 10% to 0.25% (27). A one-step reduction of
this magnitude prevents cell multiplication. Stepwise reductions, however, result in successive increases in saturation density on passage of
the cells and an increase in the size and number of transformed foci
produced in low serum concentrations. After a final reduction from
0.5% to 0.25% calf serum, there is at first only limited multiplication,
but serial passage in the latter is accompanied by increasing capacities
for multiplication and focus formation in that extremely low serum
concentration. The cell populations that had adapted to multiplication
in 0.25% serum lost that ability fairly rapidly when passaged in 10%
serum. Because the population adapted to 0.25% serum multiplied
more slowly in 10% serum than cells that had been passaged exclusively in the latter, there would be selection of any variants that could
multiply faster in 10% serum with a loss of capacity to multiply in
0.25% serum. The results indicate there is a high rate of generation of
genetic variants with different capacities for multiplication in various
serum concentrations. The NIH 3T3 cells apparently have an unusual
capacity for heritable adaptation to restrictive growth conditions because Swiss 3T3 cells do not exhibit such a capacity (28), and this
may be related to the ease with which the former cells undergo
spontaneous transformation.
The acute sensitivity of the NIH 3T3 cells to small differences in
population density is illustrated by varying subconfluent densities
over a narrow range in 2% calf serum and testing at intervals for an
increase in saturation density (29). The rate of increase in saturation
density tested every third LDP was proportional to seeding density.
Although none of the LDPs reached confluence during each passage,
there was contact between cells that increased with seeding density,
and this apparently was sufficient to account for the increase in
selectivity with population density. A critical test demonstrated that
the increase in population density rather than the increase in cell
number per se was responsible for the development of focus-forming
ability (30).
Epithelial Cells. Most human cancers are of epithelial origin, so it
is of particular interest to examine the role of selection in spontaneous
transformation of epithelial cells in culture. This was done with an
established line of diploid rat liver epithelial cells that multiplied at a
maximum rate in multicellular islands that developed even at low
density. The rate of multiplication slowed down at 1 week when the
islands became confluent with one another (31). Cells were maintained either under nonselective conditions by weekly passages just as
they reached confluence or under selective conditions by monthly
passages that included 3 weeks of constraint at confluence. Experimental cells were also treated with the mutagenic carcinogen MNNG
and cultured under selective and nonselective conditions, as were
untreated controls. The MNNG-treated and control cultures under
selective conditions started to initiate liver carcinomas on injection
into syngeneic rats after about 16 and 20 cell doublings in culture,
respectively. About five to six times as many cell doublings were
required for the cultures under nonselective conditions to become
tumorigenic, with no significant difference between the MNNGtreated and untreated cultures. The results demonstrated the powerful
accelerating role of selection in developing the tumorigenic capacity
of epithelial cells. They also showed that the appearance of tumorigenic variants occurred at almost as high a rate in untreated controls
as in cultures treated with a mutagenic carcinogen. Evidently, the
mutation rate was already high enough in the untreated cultures to
provide neoplastic variants for selection; treatment with carcinogen
added only marginally to the spontaneous rate. The fly in the ointment
of this interpretation of the results is the possibility that long-term
confluence is itself mutagenic. Whereas this possibility was once
entertained for fibroblasts (16, 17), other considerations (13, 18, 24)
eventually ruled it out, and it can be safely disregarded for the
epithelial cells. Hence, we are left with the plausible conclusion that
the rate of spontaneous variation is high in both fibroblast and epithelial cell cultures, and selection is the driving force in advancing the
cells to the neoplastic state.
Cellular Diversity as Substrate for Selection
Primary Cultures. The average mutation rate of animal cells has
been estimated to be about 1.4 ⫻ 10⫺10 per nucleotide/per cell
division, based largely on studies of cells in culture (32). The estimated rate of point mutations in the human germ-line, however, is
about 200 times higher, and this does not include small duplications,
rearrangements, or deletions (33). Mutations at micro- and minisatellites occur at 1 or more orders of magnitude higher (5). Because
mutations accumulate with time, their frequencies in tissues are higher
than their estimated rates (5, 34). More to the point regarding variation
related to neoplastic transformation is the observation that many foci
of altered cells appear in secondary cultures of baby mouse mesenchymal cells maintained in a quiescent state for 6 – 8 weeks (35).
Exposure of primary cultures to fluorescent light or oxygen gave rise
in the secondary cultures to much larger foci that continued to enlarge
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after the background cells had stopped multiplying. There was great
variation in the arrangement of altered cells between the foci that
overgrew the background in contrast to the similarity of altered cells
within a particular focus. Although altered cells did not produce
tumors in syngeneic mice, they had the morphology of transformed
cells and were considered preneoplastic (36). This was an indication
of selectable preneoplastic variants in a normal population of cells
from very young animals.
Similar foci of increased population density also appeared in primary cultures of diploid rat embryo liver epithelial cells that were held
at confluence for 1 week or longer (37). Such foci reappeared in
secondary cultures that were held at confluence. When later passages
of such diploid hepatocytes were subjected to selection under repeated
rounds of prolonged confluence, they became tumorigenic in about 4
months (31) as described above in “Epithelial Cells.” They produced
a diversity of tumor types (including hepatocellular carcinomas,
cholangiocarcinomas, hepatoblastomas, and osteogenic sarcomas),
but individual lineages yielded tumors with consistent and specific
patterns of differentiation (38). It is plausible that the densely packed
foci that appeared in the confluent primary cultures were precursors of
the cells that became tumorigenic through the accumulation of independent transforming events during the selective regime. The foci are
indicators of diversity in the growth capacity of cells in the original
cultures of the liver epithelium, just as they are in normal fibroblast
cultures (35), and probably provide the most favorable material for
progressive selection of the neoplastic state. The implication of the
diversity among spontaneously occurring foci of independent origin
among fibroblasts and the diversity of tumors produced by separate
lineages of transformed rat liver epithelia is that there were either
intrinsic differences among the cells in each culture or that the
stochastic accumulation of mutations differed among independent
transformants. In either case, transformation reveals a higher level of
variation than would otherwise be expected by visual inspection of
cultures under nonselective conditions.
Established Cell Lines. The extent of variation detected in nontransformed and transformed sublines of the Balb/3T3 line of mouse
embryo fibroblasts was so high as to be difficult to account for by
conventional mutational rates. Each of the subclones isolated from a
clone of nontransformed cells and a clone of transformed cells consistently differed from one another in a variety of morphological,
growth, tumorigenic, and biochemical properties (39, 40). Cells from
each of the subclones had a wide range of chromosome numbers, with
averages around the tetraploid number (40). The colony-forming
capacity in agar of a transformed clone was reduced 10-fold during
tumor formation by the cells, and there was wide variation in this
property among subclones from one tumor, but not from a second one
(41). Differences in morphology, colony formation in agar, and tumorigenicity arose early in subclones of a transformed clone. Secondary subclones differed within each subclone, but not as much as they
differed as a group from those derived from other subclones. Similarly
high degrees of variability were later obtained among agar colonies
derived from a clone of the Balb/3T3 cells and in subclones from
those agar colonies (13). It was tempting at the time to speculate that
the high degree of variability in growth properties within clonally
derived populations was the result of epigenetic changes, but subsequent considerations from studies on spontaneous transformation in
NIH 3T3 cells discussed below lent weight to a genetic interpretation.
Spontaneous transformation was much more regular in NIH 3T3
cells than in Balb/3T3 cells and could be efficiently measured by
counting multilayered foci of transformed cells on a monolayered
background of nontransformed cells (16). Fine features of morphology of the foci frequently allowed discrimination of variants, whereas
the size and thickness of the foci were signposts of neoplastic pro-
gression that were reflected in their tumorigenic capacity (17), as
noted previously with C3H 10T1/2 cells (21). Progression could also
be followed by the procedure of serial assays at confluence, which
revealed a sequence proceeding from no visible foci through foci of
increasing size and population density.
Once the transformability of the NIH 3T3 cells had been reduced by
frequent LDP to the extent that no dense foci appeared in a 1° assay,
populations could be grown up from relatively small starting numbers,
and the focus-forming capacities of each of many populations could
be determined in 2° assays (42). This procedure allowed rough estimates of the heterogeneity of the competence for transformation in the
original population. The results showed that a very low proportion of
cells could progress rapidly to formation of many dense foci and that
there was diversity among cells for selection under the growthconstraining conditions of the assay. As was the case for the Balb/3T3
cells, there was a wide range of variation in potential for focus
formation among subclones of the same clone, with larger differences
among subclones from different clones (43, 44). A wide range of
differences among foci was detected by quantitative computer scanning of the area and density of the foci (45). As another indicator of
variation, every cell in nontransformed populations proved to be
unique in the precise distribution of chromosomes, including marker
chromosomes (46). Therefore, chromosome variation was occurring
at a much higher rate than conventional point mutations.
Some idea of the dynamics of progression in small populations can
be obtained by seeding the cells in multiwell plates, growing them to
confluence, and assaying many of the populations at successive intervals for focus formation with an excess of nontransformed cells.
The wells were one-seventieth the area of the 21-cm2 dish used for
conventional assays, and the starting and final confluent cell numbers
were reduced in proportion (47). Each series began with the production of small light foci from cells assayed from a few of the wells. The
number of wells with cells that produced light foci increased rectilinearly with time and was followed by wells that produced foci of
increasing size and density, until all wells were producing foci by
7–10 weeks. The result suggests that a high proportion of cells can
give rise to focus formers if selective conditions are maintained long
enough.
There are several problems in calculating rates of variation from the
foregoing results. One is the heterogeneity of the cell population
undergoing transformation, which is itself shifting even under nonselective conditions, as will be considered below. A second problem is
the progressive nature of transformation. This presented difficulties
even when light, small foci were considered the first indications of
transformation. The difficulty was compounded when it was realized
that selection was occurring under the constraint of confluence even
before discrete foci appeared. This was already implicit in the observation using cells that had undergone extensive selection against
transformable cells in frequent LDP. It then required several serial
rounds of selection at confluence before foci became visible, indicating that selection was occurring without visible, discrete lesions in the
early rounds (19). Such selection of incipiently transformed cells
became apparent in a multilineage experiment with a particular batch
of cells that had enough selectable cells to produce successive increases in saturation density with each serial round of confluence
without producing foci in the early rounds (Ref. 20; Figs. 2 and 3).
The excellent agreement among parallel quadruplicate lineages left no
doubt about the accuracy of the saturation density measurements, and
their agreement with the preceding total number of cell divisions
reinforced the conclusion that early steps in transformation occurred
before focal lesions were visible.
One way to estimate of rates of variation is to start from cell
populations expanded in a known number of divisions from single
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cells and maintained under the nonselective conditions of continuous
exponential growth by frequent LDP. They could then be tested for
early signs of focus-forming capacity at intervals by assaying aliquots
of the cells in serial rounds of confluence. This was first done with 29
clones from cells newly thawed from the original, easily transformed
stock of NIH 3T3 cells (48). Most of the clones produced light foci on
assay of cells that had undergone 5 or 13 LDPs. The clones multiplied
from a seeding of 2 ⫻ 104 cells to about 5 ⫻ 105 cells per culture at
every passage or about 5 ⫻ 105 cell divisions. The rate of transformation to light focus formation would then be between 1.5 and
4 ⫻ 10⫺7 per cell division. These figures are about 10-fold higher
than the values for spontaneous transformation estimated for dense
foci in populations of C3H 10T1/2 cells (49). The difference may be
due in part to the likelihood that many of the dense foci represent
progression from light foci. The estimate for the light foci may itself
be too low because there are subliminal stages that expand selectively
and progress to transformed focus formation (20). The values would
then be higher than those characteristic of mutation at a single locus
(49) but not unexpected if mutation at each of many loci may afford
selective advantage at confluence, and if chromosomal alterations are
involved (5). The “many site” concept is consistent with the morphological diversity of spontaneously arising foci in secondary cultures of
mouse fibroblasts (36) and NIH 3T3 cells (19) and the diversity of
tumor types produced from independent lineages of early passage
diploid rat liver epithelial cells that had undergone spontaneous transformation under selective conditions (38). One must also take into
consideration the fact that different sublines derived from the original
NIH 3T3 line have widely different susceptibilities to spontaneous
transformation (18, 50), as do clones derived from the same culture
(48, 51).
Population Drift of Transformability
It has already been pointed out that the NIH 3T3 cell line becomes
less transformable if repeatedly passaged in frequent LDPs (18). To
avoid such variability, many researchers do not use cells beyond a
limited number of passages from the original frozen stock. However,
this has the disadvantage of missing important features of the transformation process that are revealed with cells of different susceptibility to transformation such as its early incipient stages and progressive nature. It is therefore of interest to be aware of the different
possible courses of evolution in populations under nonselective conditions and the possible contribution of intraclonal change in such
evolution. To this end, six clones from the original, easily transformed
stock of NIH 3T3 cells were passaged 56 times every 3 days at low
density, and aliquots of cells were tested at six different passage levels
for transformability in serial rounds of confluence (51). One of the
clones produced a few tiny, dense foci in 1° assays after the early
LDPs. Cells from these foci produced large, dense foci when reseeded
in 2° assays. After many LDPs, this clone produced only light foci in
the 1° assay that progressed to dense foci in the serial assays. Four of
the clones produced only light foci in all of the 1° assays, although
these always progressed to dense focus formation in the serial assays.
The sixth clone behaved like the preceding four when assayed at the
first five passage levels but progressed to dense focus formation in the
1° assay after the last passage. That clone behaved differently from
previous experiments with uncloned cultures, which usually decreased
in transformability after many frequent LDPs (18, 52). It was also
unexpected because most transformed populations multiply more
slowly at low density than their nontransformed progenitors (21–23,
49). The aberrant behavior of this clone serves as a reminder that all
statements about transformability are probabilistic rather than categorical.
Clonal Expansion Advances Transformation
It is generally accepted that most human tumors arise from the
expansion of a single clone (53–56). More than one cell in a population may give rise under selective conditions to cells at various stages
of transformation in culture, but one of these subpopulations has a
higher selective advantage than the others and eventually predominates (48, 57). Given the difference in transformability of clones
derived from the same culture, it was of interest to know what clonal
type would dominate in spontaneous transformation. This was fairly
obvious in an easily transformed population containing some clones
that became fully transformed much faster than the others (48) but
was less apparent in a cell population that was relatively refractory to
transformation. One such uncloned population exhibited no increase
in saturation density or any sign of focus formation in five rounds of
selection at confluence and only the barest suggestion of foci in the
sixth round (58). Parallel assays of clones from that population revealed that some of them produced well-defined foci beginning in the
third and fourth round of confluence. This seemed surprising at first
glance because the polyclonal parental population must have had
thousands of cells that could give rise to the neoplastically productive
clones. However, only one-fifth of the clones were highly productive
of foci, and then only after three or four rounds of confluence. The
development of foci depends on the number of responsive cells
present under the selective condition of confluence. The polyclonal
parental population would have only one-fifth as many responsive
cells at confluence as the neoplastically productive clones (which
constituted only one-fifth of the total population) and therefore would
not be expected to exhibit frank transformation until it was subjected
to selective conditions five times longer than the clones, i.e., after
15–20 rounds of confluence. The experiment illustrates the importance of expansion of neoplastically productive clones in the genesis
of transformation and, conversely, the importance of maintaining a
balanced distribution of clones in preventing transformation. The
latter point is reinforced by the evidence that normal cells surrounding
transformed cells can, in some combinations, suppress expansion of
the transformed cell (59 – 62).
The Nature of Transformation
In the early stages of systematic analysis of spontaneous transformation, it seemed that an epigenetic mechanism was at least as
convincing as a genetic explanation of the phenomenon (16, 17). The
fact that transformation was most frequently encountered when the
cell population as a whole was quiescent seemed to argue against a
mutational origin, which is conventionally correlated with cell division. Once a cell population was obtained that required several rounds
of confluence for visible transformation (18), there seemed to be no
evidence for selection in the early stages because there was nothing
visible to select. Then there were indications that the transformation
was reversible (17, 63) and that even tumors of varied origin in
animals and humans would revert to normal under appropriate conditions (64 – 69). Later, however, it became apparent that cells were
present that had a selective advantage at confluence but did not
produce visible lesions (20). Multiplication and selection could occur
in a minority population while there was no increase or even a
decrease in the total number of cells. Apparent reversal could occur in
LDP when there was contamination of a transformed clone with a few
nontransformed cells because the latter usually multiplied faster at
low densities than the former (23). Indeed, this raised the possibility
that there was selection for reverted cells under optimal growth
conditions. Reversion (as well as progression) could occur at a much
higher rate than the common estimates of point mutations (32) if the
chromosomal alterations so common in established cell lines play a
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significant role in transformation, as they apparently do in human
tumor development (70). However, when rigorous precautions were
taken to isolate pure populations of transformed cells, the transformation proved to be irreversible (71). The sum of these qualifications
and observations tipped the balance in favor of a mutational origin of
the transformation, but one that usually occurred in a progressive
manner in several steps.
The mutational view was reinforced by the finding that the few
cells in a Swiss 3T3 line that could multiply in a sharply reduced
concentration of serum were unable to maintain that capacity in
subculture (72), unlike the NIH 3T3 cells in various stages of progression. The idea that nonspecific heritable damage to cells might
increase the chance of transformation (23) was weakened by the
failure of heritable damage induced by methotrexate to increase
transformation of NIH 3T3 cells (73). It seems most likely therefore
that spontaneous transformation is driven by selection of genetic
variants produced in culture at the ordinary rate. Selection is the
critical condition for the progressive emergence of those variants to
the fully transformed state. The epigenetic components of transformation therefore are those selective conditions—suspension in soft
agar, low serum concentration, fetal serum with low growth factor
activity, and contact inhibition at confluence—that drive progressive
transformation. As noted below, however, selective conditions in the
organism are likely to be quite different than those in cell culture.
The Role of Selection in Experimental and Human Cancer
As noted earlier, interest in the role of selection in human neoplasia
was heightened by recent computer models of colorectal cancer that
indicated that selection is the driving force in the origin of most cases
of this condition and probably of other human cancers as well (1, 2).
However, there was already considerable evidence of a prominent role
for selection in experimental cancer of animals. In cell transformation
in culture, contact inhibition of confluence is the most frequently used
selective condition; however, it cannot play that role in vivo, where
epithelial cells are always in direct contact with each other. Studies of
liver carcinogenesis in rodents reveal evidence of selection. Tens of
thousands of microscopic foci of enzyme-altered cells appear in rat
liver soon after carcinogenic treatment begins (74). Up to 10% of
them progress to macroscopic nodules, most of which remodel into
normal-looking structures (68), but a few progress to carcinoma (75).
Careful examination of livers in untreated rats reveals the presence of
small numbers of enzyme-altered microscopic foci that increase
sharply with age to a maximum of about 100 (76, 77). These foci
resemble the many thousands seen after carcinogenic treatment. Treatment with promoter alone, which is by definition nonmutagenic,
raises the number of foci to about 1000 (78). A much higher percentage of cells in the enzyme-altered foci of untreated rats are synthesizing DNA than cells in the surrounding areas (77), and this advantage persists in rats treated with promoter alone (78). The presence of
the foci in normal rat liver and their increase with promoter treatment
implies that there are incipient foci in the normal liver that are selected
by the promoter because of their growth advantage. Full carcinogenic
treatment either creates more of them by inducing mutations or is an
even more efficient selective agent than the promoters. Farber (79) has
proposed a resistant hepatocyte model of liver carcinogenesis in
which cells in foci and nodules are selected while the surrounding
cells are suppressed by the carcinogenic treatment.
Contrary to the conventional wisdom that promoters do not produce
tumors of the skin, long-term repetitive treatment of SENCAR mice
with promoters alone produced occasional papillomas and carcinomas
(80). Most of them had a mutation in the same codon of the c-Ha-ras
oncogene, suggesting that the promoters selectively induced expan-
sion of epidermal cells already bearing this mutation. There are also
a number of instances in which carcinogens apparently selected cells
with spontaneous ras mutations for tumor growth (81). More recently,
Ha-ras 1 mutations were found in small patches of mammary epithelium in young untreated Fisher female rats (82). Treatment with
N-nitroso-N-methylurea resulted in mammary tumors, ⬎90% of
which carried the Ha-ras 1 mutation. This mutation was rarely seen in
mammary tumors induced by other carcinogens. The implication is
that N-nitroso-N-methylurea specifically selected for growth of cells
bearing the preexisting ras mutation. The selective multiplication of
ras-activated cells would increase the opportunity for additional mutations and drive progression to malignancy.
There are a number of examples in experimental animals in which
chronic endocrine imbalance stimulates persistent cell multiplication
in specific tissue, leading eventually to tumor formation (65). The
hormone imbalance is not directly mutagenic but presumably fosters
the selective growth of cells with a growth advantage in that imbalance. In some of these endocrine disturbances, correction of the
hormone imbalance results in disappearance of the tumors, including
metastases that developed during the imbalance. This indicates that
persistence of the selective environment is necessary for continued
neoplastic expression of the altered cells.
Homeostasis of epithelial tissues, such as epidermis and intestinal
mucosa, is maintained by topographical features of cell multiplication,
differentiation, and apoptosis. Continuous multiplication occurs in the
basal layer of the epidermis. Cells that move into a higher layer lose
the capacity to multiply. Similarly, stem cell multiplication occurs in
the lower epithelial layers of colorectal crypts and continues in transit
cells ascending toward the lumen (83). The cells stop multiplying in
the upper one-third of the crypt and are extruded into the lumen. In
both tissues, treatment with carcinogen produces diffuse, persistent
hyperplasia in which cells multiply in previously restricted sites (84,
85). The first sign of true neoplasia is the appearance of nodular
growth in the form of papillomas of the skin and polyps of the colon.
Selective growth in human colorectal carcinogenesis is usually driven
by mutations in both alleles of the APC gene, which results in
dysplastic polyps known as adenomas (1, 86). Colorectal carcinomas
can also arise, albeit less frequently, by alteration of the microenvironment that results from excessive proliferation of the underlying
stroma in juvenile polyposis syndrome (87). Disruption of the normal
architecture of the tissue in both these cases produces a microenvironment that interferes with the orderly regulation of growth. The
increased multiplication facilitates genetic changes that drive progression to carcinoma.
Another indication that breakdown in tissue structure provides a
selective environment for tumor development comes from experiments in which cells were physically dissociated from one another.
Mammary epithelium was removed from young C3H mice, dissociated enzymatically, and reinoculated into the cleared fat pad of young
syngeneic mice (88). This procedure greatly accelerated the development of hyperplastic alveolar nodules, which are a precursor to
carcinomas and only appear after a protracted period of time in
undisturbed mammary glands. If cells of the extirpated epithelium
were not dissociated from one another before reinoculation, there was
much less acceleration of tumor development (89). Hence, the normal
architecture of the mammary gland is a restraint against neoplastic
growth.
Tissue microenvironment is also affected by age. Inoculation of rat
hepatocarcinoma cells into the liver of syngeneic rats produces a
tumor much more readily in old rats than in young rats (90). Multiplication of normal crypt cells of the intestine slows down in aging
mice, and the time of onset of DNA synthesis in the cells becomes
more heterogeneous (91, 92), suggesting the accumulation of damage
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to DNA. It is not unlikely that this decreases the capacity of the
mucosa to regulate the growth of rogue clones and increases the
likelihood of tumor formation. Radiation damage to the lungs of mice
increases the number of metastatic lesions produced there by i.v.
inoculated tumor cells (93, 94). Preirradiation of the cleared mammary fat pad of mice increases the frequency of tumor formation by
neoplastic epithelium grafted into that site (95).
The implication of the mammary grafting experiment is that the
state of stromal tissue plays an important role in epithelial tumor
development. It has, in fact, been known for some time that epithelialmesenchymal interactions have a strong influence on tumor development (96). The unit responding neoplastically to polyoma virus infection was the complex of epithelium and mesenchyme, with neither
responding alone. Many related findings made in recent years were
recently summarized in a report that fibroblasts associated with prostatic carcinomas direct neoplastic progression of initiated but nontumorigenic prostatic epithelium (97). Normal prostatic fibroblasts did
not have this effect. Therefore, in some cases, normal fibroblasts are
required for tumor growth (96), and in others, they hold it in check
(97), but the importance of tissue interactions in tumorigenesis is
inescapable.
Evidence for the importance of the local environment in tumor
development is not restricted to experimental animals. Metastases
were found in the cervical lymph nodes in patients with no primary
tumors in the mucosa of the upper aerodigestive tract (98). However,
identical genetic lesions were found in the lymph node metastases and
in defined areas of the histologically normal mucosa that were common sites of primary tumors. Some of those normal sites eventually
did develop into carcinomas. The results indicate that the normal
mucosa was able to regulate the growth of preneoplastic epithelial
cells that were already forming metastases in the microenvironment of
the cervical lymph nodes.
The foregoing discussion emphasizes the role of altered tissue
topography and microenvironment in selecting rogue clones for tumor
growth. The APC mutations of the colorectal epithelium are apparently sufficient to allow selective growth of the altered cells into early
tumors without additional mutations or microenvironmental changes
(99). Analysis of mouse aggregation chimeras shows that the action of
the APC mutations and that of modifier genes are localized within the
cell lineage that gives rise to intestinal tumors (100, 101). This does
not rule out a role for microenvironmental effects in determining the
growth of intestinal tumors because the stem cells of the human small
intestine multiply even faster than colorectal stem cells (102) and are
therefore likely to have at least as many APC mutations, but they very
rarely form tumors. This disparity may be related to the difference in
the distribution of dividing cells in the crypts (102) and in the
architecture of the two tissues: the epithelial crypt cells of the small
intestine ascend into elongated villi that extend into the lumen,
whereas those of the large intestine are extruded directly from the
crypts into the lumen (103). Whatever the explanation, it seems that
selective growth can arise (a) in a cellularly autonomous fashion by
mutation in the prospective cancer cell, (b) by differences in the
regulatory microenvironment, or (c) by some combination of both.
Mutation Frequency in Normal Tissues
It is axiomatic that the effectiveness of selection for tumor formation depends on the frequency of mutation in tissues. Simpson (5) has
surveyed the literature on the frequency of somatic mutations in solid
tissues of humans and concluded that it can account for multistep
carcinogenesis. For example, the frequency of somatic mutations in
the HPRT gene of normal kidney cells of men rises from 5 ⫻ 10⫺5 in
the first decade of life to 2.5 ⫻ 10⫺4 beyond the age of 70 years (34).
The APC tumor suppressor gene is considerably larger than the HPRT
gene, and the estimated frequency of inactivating mutations of this
gene is 1.7 ⫻ 10⫺3 per cell (5). Given the square of this figure for the
frequency of biallelic APC mutations, there may be many colorectal
cells in the colon of aging people that carry biallelic mutations in this
tumor suppressor gene and therefore have the potential for tumor
growth. In this scenario, there is no need to invoke higher mutation
frequencies to account for multistep human carcinogenesis. Indeed,
there is the implicit suggestion that many such mutated cells are held
in check in normal tissues. In fact, it was recently reported that there
are about 11,000 mutations of inter-simple sequence repeats in polyps
and carcinomas of the human colon (104). This suggests there may be
many mutations of this type in normal crypts of the colon, but these
would be hard to detect because of their occurrence in single, unexpanded crypts surrounded by a large excess of crypts without the same
mutations in the normal tissue sample.
Continuing along this line, clones have been isolated from histologically normal human breast by dissecting ducts and terminal ductal-lobular units (105). Nine highly informative microsatellite markers
were examined, half of which were at chromosomal regions involved
in breast cancer. At least one genetic abnormality was found in half
the women examined with a weighting toward mutations known to be
involved in breast cancer, but there was no association with histological abnormality, nor did genetic abnormalities necessarily lead to
cancer many years later (106). These considerations indicate the need
for additional genetic changes in the lesion-bearing clones and/or
microenvironmental changes that permit selective growth of the
clones for neoplastic development to occur.
General Considerations and Future Directions
The prominence of selection in tumor development in a sense is an
extension of findings in experimental embryology that began in earnest with Spemann’s discovery (107) of the role of the organizer in
early frog development. An intriguing offshoot was the demonstration
that differentiation depended on tissue fragments larger than a minimal size (108). Whereas the fate of large blocs of tissue may be
strictly determined, that of individual cells in those blocs is not (109).
Such observations have been formalized in the concept that there can
be regularity in the large where there is heterogeneity in the small or
“order above heterogeneity” (110). This concept was developed on the
basis of heterogeneity of the atomic and molecular constituents of
cells that are ordered by higher levels of organization from the cell to
the organism. If genetic changes of DNA are inserted in this hierarchy, one might infer that great genetic diversity is generated in the
growth and development of somatic cells but is usually not expressed
in the integrated architecture of a tissue. A breakdown in the ordered
state can come from the accumulation of multiple genetic changes in
a prospective tumor cell or in the ordering capacity of its microenvironment or a combination of the two. Both aspects of control may be
weakened by the accumulation of somatic mutations in the aging
process. Most of the emphasis in contemporary cancer research has
been on finding genetic changes in tumor cells, but this effort needs
to be extended to clones in normal tissues to provide a broader
foundation for understanding normal regulation and its breakdown in
malignancy.
The normal tissue controls commonly used as a baseline for identifying genetic changes in tumors involve many clones. Random
mutations in the DNA of any clone are swamped out by the DNA
from all of the other clones in the tissue sample. Techniques have to
be applied to isolate the DNA from clones or otherwise identify clonal
mutations, i.e., immunocytochemistry, clonal selection, and so forth.
The continuous improvement of methods for characterizing small
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amounts of DNA, e.g., PCR, should facilitate the task. It should then
be possible to evaluate the relative contributions to tumor growth of
mutation in a potential cancer cell and the regulatory capacity of its
microenvironment.
Addendum
Haddow (111) first adduced a role for selection in tumor development from the contrast between the inhibition of spontaneous tumor
growth by carcinogenic polycyclic aromatic hydrocarbons and their
failure to inhibit the growth of tumors induced by the same and other
compounds. Recently, evidence was presented for the thesis that
selection is a critical factor in determining the mutations found in
human cancer (112). Additional evidence indicates that physiological
stresses aggravated by carcinogens in tobacco smoke select endogenous mutations to drive the development of lung cancer in humans
(113). Loss of function mutations in the p53 tumor suppressor gene
select, under stressful conditions, for cells which would otherwise be
eliminated by apoptosis (114).
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Selected Cell and Selective Microenvironment in Neoplastic
Development
Harry Rubin
Cancer Res 2001;61:799-807.
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