[Cancer Research 61, 799-807, February 1, 2001]
© 2001 American Association for Cancer Research
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
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ABSTRACT
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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.
"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."
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Introduction
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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.
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Selection in Transformation of Primary Cultures
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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.
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Selection in Transformation of Established Cell Lines
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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 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, 22, 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 mutations that would drive further selection,
culminating in the formation of distinctive transformed foci, each
derived from a single cell.
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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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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 selection
operates on cells before they produce clearly visible, discrete foci,
thus recalling the incipient state of neoplasia described by Foulds
(25
, 26) .
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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.
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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 MNNG-treated 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.
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Cellular Diversity as Substrate for Selection
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Primary Cultures.
The average mutation rate of animal cells has been estimated to be
about 1.4 x 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 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 progression
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 growth-constraining 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 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 x 104 cells to about 5 x 105 cells per culture at every passage or about
5 x 105 cell divisions. The rate
of transformation to light focus formation would then be between 1.5
and 4 x 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)
.
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Population Drift of Transformability
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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, 22, 23
, 49)
. The aberrant behavior of this clone serves
as a reminder that all statements about transformability are
probabilistic rather than categorical.
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Clonal Expansion Advances Transformation
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It is generally accepted that most human tumors arise from the
expansion of a single clone (53, 54, 55, 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, 60, 61, 62)
.
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The Nature of Transformation
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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, 65, 66, 67, 68, 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 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 expansion 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 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 epithelial-mesenchymal
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 x 10-5 in the first decade
of life to 2.5 x 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 x 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 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)
.
|
FOOTNOTES
|
|---|
1 To whom requests for reprints should be
addressed. Phone: (510) 642-6617; Fax: (510) 643-6791; E-mail: hrubin{at}uclink4.berkeley.edu 
2 The abbreviations used are: LDP, low density
passage; MNNG,
N'-nitro-N-nitrosoguanidine.
Received 7/21/00.
Accepted 11/21/00.
|
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Top
ABSTRACT
Introduction
; 5% calf serum, •; and 10% calf
serum, 
. The open symbols (
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.