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Tumor Biology |
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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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the studies of the genetics of human cancer have highlighted the role of selection, they provide little information on the dynamics of the process. However, studies of spontaneous neoplastic transformation in cell culture provide considerable insight on the subject. The first indication that selection plays a role in spontaneous transformation came from studies of the adaptation of mouse embryo fibroblasts to cell culture, which revealed that there were large increases in saturation density after multiple passages of cells at high density (12) . Because the multiplication of normal cells is inhibited by contact at high population density, it appeared that there was selection of variants that overcome that inhibition, a characteristic of neoplastic cells. The role of selection was supported by the finding that mouse embryo fibroblasts passaged at high density gained the capacity to produce sarcomas in syngeneic mice within 3 months, whereas those passaged at low density did not become tumorigenic through the entire 9 months of the study (13) . Additional support for selection as the driving force in spontaneous transformation came from findings that it was produced in established lines of mouse fibroblasts by procedures that selectively inhibit the multiplication of normal cells, such as suspension in soft agar (14) , long-term contact inhibition in confluent cultures (15) , or reduced growth rate in either low serum concentration (16) or in fetal serum with suboptimal growth-supporting activity (15 , 17) .
All of the foregoing studies used fibroblasts, but most human cancers are of epithelial origin (18) . It is therefore of particular significance for human cancer that selective conditions were found to promote transformation in epithelial cells. Diploid rat liver epithelial cells kept in a stationary state at confluence for 3 weeks between every monthly passage gained the capacity in only 20 cell divisions to produce liver carcinomas in syngeneic rats, whereas those passaged weekly before they became confluent did not produce tumors until they had undergone about 6 times as many divisions (19) . A significant sidelight occurred when the liver epithelial cells were treated with a mutagenic carcinogen before being cultured under selective and nonselective conditions. The treated cells became tumorigenic under both regimes at about the same disparate times as they had in spontaneous transformation, indicating that selection was the dominant force in transforming carcinogen-treated cells as well. Indeed, the carcinogen seemed to add little to the spontaneous variation that provided the altered cells for selection. There was no correlation between aneuploidy and tumorigenicity in the spontaneously transformed populations: populations of aneuploid cells often were nontumorigenic. However, there was a correlation in the carcinogen-treated cells, indicating that the carcinogen treatment produced a specific type of aneuploidy that was associated with carcinogenesis.
The question of selection versus genetic instability arose again in spontaneous transformation of the NIH 3T3 line of mouse fibroblasts. Cells from the original transformation-sensitive stock of NIH 3T3 cells gave rise to clones among which there was a small minority that started to produce a few dense transformed foci at confluence sooner than the polyclonal population from which the clones were derived (20) . When six of the clones were cocultured before the first round of confluence, they produced many fewer dense foci in subsequent rounds than expected from averaging the number that occurred in parallel cultures of the individual clones (21) . It was proposed that the individual clones were genetically unstable, and intimate contact with other clones stabilized them. We pursued this proposal further in the present study using clones of a subline of NIH 3T3 cells that is relatively refractory to spontaneous transformation. We report that a minority of the clones produced well-defined transformed foci well before there was any sign of transformation in the uncloned parental culture. A quantitative analysis of the results shows that the disparity can be fully accounted for by the much smaller number of transformable cells from the susceptible clones in the polyclonal parental population at confluence than there are in each susceptible clone at confluence. Simple numerical accounting thus eliminates the need for the arbitrary assumption of genetic destabilization by clonal isolation to explain preferential transformation in the minority of clones that were susceptible.
This conclusion, of course, concurs with William of Ockham’s maxim, "Multiplicity ought not to be posited without necessity," otherwise known as Occam’s Razor.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cells were cloned by seeding an average of one cell/well in 96-well
plates (Falcon 3072) with growth medium MCDB 402 plus 10% CS vol/vol.
The approximate number of cells/well was monitored under the microscope
and recorded at 2, 4, 5, and 6 days. Wells containing more than one
colony were excluded. Clones were designated by the coordinate position
of the well they occupied. Twenty-seven clones were harvested by
trypsinization at 5, 6, and 7 days and divided into five groups, as
shown in Figs. 1
-3, depending on their initial growth rates as estimated from the cell
number in the wells. The clones were expanded in
21-cm2 dishes and subcultured five times in
LDPs of 5 x 103 cells/dish
every 4 days. The uncloned cells were maintained in parallel with the
clones by the same LDPs. The growth rate of each clone and of the
uncloned culture was estimated by the cell yield at each passage. At
the fifth passage, an aliquot of the cells was used to initiate the
first set of six serial assays at confluence for transformation, and
the remaining cells were used to continue the LDPs. At the 13th LDP, a
second set of four serial transformation assays was initiated.
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The best description of their morphology comes from viewing photographs
of selected clones and the uncloned culture. Those confluent assay
cultures that were not trypsinized for passage or growth rate
determinations were fixed with Bouin’s reagent and stained with 4%
Giemsa buffered at pH 7.0 to determine the extent of transformed focus
formation. As is documented with photographs (e.g., Fig. 4
),
the uncloned culture produced no discernible foci in the first set of
serial assays, but many of the clones did. However, the foci produced
by the A' clones did not reach the large size and high cell density of
those produced by the transformation-sensitive SA' cells in our
previous studies (20
, 23
, 25)
. The scale used to
characterize the degree of morphological transformation is shown in
Fig. 6
. Where the transformation was particularly strong, it resulted
in a marked increase in saturation density of the assay culture.
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RESULTS |
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shows the growth rates of the cells in every clone during the
first four LDPs arranged according to group number. The clones of group
1 were relatively uniform with a group-averaged growth rate of
1.85 ± 0.08 PD/D (the "±" part is "the average
of the absolute deviations of data points from their mean"). Four of
the group 2 clones multiplied relatively rapidly (averaged
1.86 ± 0.14 PD/D), but two of them (6 h averaged
1.27 ± 0.07 PD/D, and 8 h averaged 1.57 ± 0.03 PD/D) were distinctly slower than the rest. The uncloned
A' culture was kept with group 2 and had a consistently high growth
rate (averaged 1.90 ± 0.09 PD/D). There was a wide
spread in the growth rates of groups 3, 4, and 5 (1.64 ± 0.17 PD/D, 1.74 ± 0.14 PD/D, and 1.49 ± 0.27 PD/D, respectively) with group 5 exhibiting the slowest
growers. The rank order of growth rates of individual clones in each
group was mostly consistent throughout the four passages, indicating
that they represented heritable differences between the clones.
Reduction in Growth Rates of Clones Subcultured after Serial Assays
at Confluence.
Table 1
shows the average growth rates for each group of clones as well as the
uncloned culture for the LDPs and for cells recovered from the first
two serial assays. Experimental cells of all groups showed a decrease
in growth rate after the assays at confluence despite the fact that
they were given a 2-day passage at low density to recover from the
constraint of confluence before passage for growth rate determination.
The extent of reduction after the 1° assay varied from 18 to 25% for
the clones, and 15% for the uncloned culture. The relative reduction
was apparently less after the 2° assay. However, a close inspection
of the data shows there was an anomalous drop in the growth rate of the
LDP controls of some clones rather than a rise in the growth rates of
their experimental counterparts. The results were consistent with the
idea that the prolonged period of constraint at confluence inflicts
heritable damage on the cells (23
, 26)
.
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shows the relative growth rates of groups 2 and 5 through the 5°
assay at confluence. With the exception of clone 8a of group 2 and
clones 8b and 5c of group 5, all of the clones and the uncloned culture
exhibited reduced growth rates after assay at confluence. However, the
heritable damage implied by these results showed no correlation with
the extent of transformation described below, particularly in the case
of the uncloned culture that showed no evidence whatever of
transformation in the first five assays at confluence.
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shows the saturation densities after each of the six assays of the
first set. The uncloned culture (dotted line in group 2) had
a higher saturation density (6 x 105
) than any clone in the 1° assay, but it
dropped to half that value in the 2° assay and remained at that low
level for all of the subsequent assays. It produced no foci up to the
6° assay, when some very faint foci appeared (Fig. 4)
. Clone 1a of group 1 showed a steady increase in saturation density
between assays 3° and 6° with a final value above
106 cells/culture in the final assay. This was
consistent with the appearance of many small foci in clone 1a beginning
in the 3° assay. Clone 10c of group 2 exhibited a marked increase in
saturation density between the 5° and 6° assay, when it went from
producing a small number to a large number of moderately dense foci.
Clone 9g of group 2 also increased significantly in saturation density
in the 6° assay as well as in the number of moderately dense foci.
Clone 6h displayed a more modest increase in saturation density in the
6° assay and had a large number of foci, but they were lighter than
those of 10c and 9g. In group 3, clone 2c increased steadily in
saturation density between assays 3° and 6° just as clone 1a of
group 1 had. This was in keeping with a large increase in the number of
moderately dense foci. The only clone in groups 4 and 5 to show a
significant increase in saturation density was clone 11a. It had
already produced a few light foci in the 1° assay. These became
denser and larger in the 3° assay and increased in number in the
4°, 5°, and 6° assays, which had increases in saturation density.
Fig. 3
, bottom right, also shows the average and SE of
saturation densities for all of the clones that began to exceed that of
the uncloned culture in the 4° assay. Whereas the saturation density
measurements of the late assays showed prominent increases for five of
the clones with no increase for the uncloned culture, it was evident by
inspection that many clones were producing visible foci that did not
result in saturation density increases. An elementary way to document
this, which captures the essence of the phenomenon that more
sophisticated measurements cannot, was through photographs of the
culture dishes. These included cultures from the first set of six
serial assays and cultures from a second set of four serial assays as
described below.
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illustrates progressive transformation in 2 clones
(1a and 2e) and its absence in the uncloned A' parental cells spanning
six serial assays of the first series. Clone 1a was negative in the
1° assay, and began showing light foci in the 3° assay (not shown);
the light foci became confluent with one another in the 4° assay, and
progressed to small, dense foci on a confluent background of light foci
in the 6° assay. Clone 2e was also negative in the 1° assay but
began to produce light foci in the 2° assay (not shown) that
progressed to discrete, moderately dense foci in the 4° assay and to
dense foci on a confluent background of lighter foci in the 6° assay.
The background of light foci was not as thick as that of clone 1a in
the 6° assay, which was reflected in the higher saturation density of
the latter (Fig. 3)
. The uncloned A' cells remained completely negative
through the 5° assay but exhibited scattered, small, light foci in
the 6° assay. The results indicate that the diverse cells of the
uncloned parental culture, which must have contained many cells similar
to those of clones 1a and 2e, underwent much less transformation than
those clones.
We entertained the possibility that some transformation was occurring
in the uncloned culture but was not expressed because of
growth-suppressing interactions among the diverse cells of this
polyclonal mixture that did not occur in the more homogeneous clones.
To investigate this possibility, the individual clones and the uncloned
parental cells were diluted for the 6° assay, and
104
cells were seeded together with a 10-fold
excess of 105
A' LDP cells to form a confluent
nontransformed background for the display of foci from the cloned
cells. Representative results are illustrated in Fig. 4B
, in
which clones 2e and 10c produce distinct transformed foci; but the 6°
assay of A' cells shows no evidence of focus formation on a background
of A' LDP cells. The 10c clone had produced small numbers of light foci
with 105
cells in the 4° and 5° assay, but
did not exhibit a significant increase in saturation density until the
6° assay, in which even 104
cells produced a
large number of foci. A culture of the LDP A' cells that formed the
background for the 6° assay cells is also shown. It is evident that
foci appeared in the 6° assay of the clones on the same background on
which the 6° assay of the uncloned population failed to produce foci.
This provides unequivocal evidence that the events that underlie
transformation simply had not occurred in the uncloned culture to
nearly the same extent as they had in some of its derivative clones.
We then examined the effect of extending the period of confluence to
determine whether foci would develop in cultures that were negative in
the standard 14-day assay period and also to reveal expansion or
progression of foci that were already apparent at 14 days. The extended
assay was done with cells from the second set of serial assays and
representative results can be seen in Fig. 5
. The foci present in clone 2e at 14 days increased in size at 28 days,
indicating the selective growth advantage of cells in the foci. Clone
4b exhibited no foci at 14 days, but a few moderately dense foci and
many small, light ones could be seen at 28 days, indicating either slow
growth of the transformed cells in this clone or delayed onset of
transformation. The uncloned A' cells failed to display any foci at
either 14 days or 28 days. The clone 2c cultures at the
bottom of Fig. 5
were from a later assay of the second set
and showed development from light to dense foci between 14 and 28 days.
The results show the diversity of dynamics of focus formation among the
clones and reinforce the evidence of greatly reduced transformation in
the uncloned population relative to some of its clones.
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. The scale of transformation is based on the seeding of
105
cells in each assay but was elaborated in the
later assays by seeding 104
cells with an excess
(105
) of nontransformed cells from the LDPs of
the uncloned A' cells. In those cases which included assays for
104
cells, the classification in the relatively
strong (++) and moderate (+) categories for 105
cells was reinforced by the appearance of discrete foci in the assay of
104
cells, which were not seen in the weak (±)
category. The graphs in Fig. 7
show an increase in the number of transformed clones in each category
with successive serial assays. About half of the clones had
moderate-to-relatively strong transformation in the last two assays of
the first series, whereas the uncloned A' cells were negative through
assay 5° and only weakly transformed in assay 6°.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 6
.) Hence, in the regular seeding of 105
cells of the uncloned parental population in the assay for focus
formation, there would be at least 2 x 104
cells capable of undergoing and clearly
exhibiting transformation if they were cultured as isolated clones, yet
no clearly identified transformed foci were detected in six serial
assays at confluence. The absence of such foci in the polyclonal
parental population indicates that some feature of the clonal mixture
reduced the number of genetic changes/culture that underlie the
transformation seen in cultures of the isolated clones. This conclusion
is augmented by the finding of large increases in saturation density of
a number of clones with successive rounds of confluence when there was
no such increase in the polyclonal population (Fig. 3)
. The failure of
focus formation in the polyclonal population persisted even when the
incubation period of the assay was increased from 14 to 28 days,
whereas the foci initiated in the clonal cultures were
increasing in size and number.
In isolating clones and expanding them to large populations in the
selective assay at confluence for transformed foci, we increase the
number of cells originating from a single cell in comparison with the
number of clonally related cells in a polyclonal population. That is,
the ratio of cell numbers in any particular clone at saturation density
to the number of phenotypically similar cells in the polyclonal
population is inversely proportional to the fraction of those clones in
the polyclonal population. If we take as a class of clones, for
example, those which eventually produce well-defined ++ transformed
foci (Fig. 6)
, an isolated clone of that class will reach confluence
with a much larger number of potential focus formers than the total
number of similar cells in the polyclonal population, in inverse
proportion to the fraction of ++ focus-forming clones among all clones
in the latter population. The smaller the fraction, the larger is the
differential. There were 5 of 27 clones that consistently produced ++
foci in the last three assays of the first set, or roughly one-fifth
the total number of clones. Therefore, the total number of ++ focus
producers in the polyclonal population at confluence will be about
one-fifth of the number in each of the isolated ++ clones at
confluence. The saturation density of each of the cloned and uncloned
cultures remains roughly constant between 2 and 4 x 105
cells during the first few assays (Fig. 3)
.
Because the production of foci is dependent on the number of
focus-forming cells at confluence, the isolated clones with the
potential for producing ++ foci would be expected to display such foci
in a much earlier assay than the polyclonal population. Well-defined
focus production first appears in two clones in the 3° assay of the
first set, and reaches a plateau of five consistent focus producers in
the 4° assay. The failure of such foci to appear in the polyclonal
population in any of the six assays of the first set (nor in any assay
of the second set) follows from the 5-fold reduction in number of
potential ++ cells in that population compared with the number in each
isolated clone. According to this crude estimate, it would take at
least 15 serial assays of the polyclonal population to produce foci of
the ++ type. A similar calculation can be made for the combined number
of + and ++ focus-producing clones, which constitute about
half of the total number of clones, e.g., if there had been
a large number of polyclonal cultures, and the number of serial assays
extended beyond six, a fraction of them might be expected to exhibit
foci by the 7° assay. If we include the somewhat marginal and
sometimes transient ± focus-forming clones in the
total, which reaches about half its maximum at about the 3° assay, it
is not surprising that some weak foci appear in the polyclonal
population by the 6° assay. Similar analysis can be applied to the
second set of assays in Fig. 7
. Therefore, the relative ease with which
the clonal populations are transformed compared with the polyclonal
parental population can be explained simply in terms of clonal
diversity in the latter.
In earlier papers using the original transformation-sensitive line that produced large, dense foci (20 , 21) , and particularly where six isolated clones were compared with a mixture of the six clones, we found that some of the isolated clones progressed to dense focus formation before any were seen in the mixture (21) . This was interpreted by assuming that isolated clones were genetically unstable at confluence, and that they were stabilized by metabolic cooperation with one another in the mixture, thereby preventing early steps in progression. The simpler explanation is that only three of the six clones progressed to dense focus formation in the 2° assay and did so at different rates. According to the inverse proportionality between a cloned and uncloned population in the number of potential focus-formers established above, there would be a 2-fold reduction of potential focus-formers in the mixture with a proportionate delay in focus formation to assays beyond those done in the experiment.
Because the argument of clonal diversity fully accounts for the paucity
of foci in the polyclonal parental culture, the previous hypothesis of
clonal destabilization is unnecessary. In fact, there is no independent
verification that clonal isolation results in an increase in the rate
of transformation-related variation. This possibility had been
considered (20
, 21)
because transformation was frequently
associated with a heritable reduction in the growth rate of cells at
low population density (26, 27, 28)
, which could be ascribed
to genetic damage with accompanying mutations. However, selection of
progressively transformed cells by LDP in low CS concentration
(16)
or fetal bovine serum (15
, 17) does not
result in a reduction in growth rate. A complementary negative finding
is that the heritable reduction in growth rate induced by methotrexate
treatment of NIH 3T3 cells does not produce transformation
(29)
. Perhaps more cogently, treatment of diploid liver
epithelial cells with a mutagenic carcinogen only marginally increased
their spontaneous rate of transformation under selective conditions and
had no significant effect on the rate of transformation under
nonselective conditions (19)
. Whereas we cannot exclude a
role for genetic destabilization in transformation for all conditions,
there is no reason to postulate such a role in the present case because
the preferential transformation of minority clones can be fully
accounted for by the selection and expansion of susceptible cells.
Additional evidence against the destabilizing effect of clonal
isolation is the existence of some clones even in the
transformation-sensitive subline of NIH 3T3 cells that fail to
transform in many repeated rounds of selection (25)
as
well as the many that show no transformation or only marginal effects
in the present experiments (Fig. 7)
.
The conditions in culture for selective growth of cells in various stages of transformation are those which preferentially inhibit multiplication of nontransformed cells. We achieved artificial selection in the present paper by isolating and expanding clones, a few of which were particularly susceptible to confluence-mediated transformation. The contact inhibition of confluent cultures was also used to drive selection of spontaneous transformants in a line of diploid liver epithelial cells (19) . Because all epithelial cells in the organism are in direct contact with their homotypic neighbors, confluence per se does not act as a selective force for carcinoma development in vivo. In the case of colorectal cancer in humans, biallelic mutations at the APC locus apparently confer selective advantage on stem cells in the colonic crypt, which allows them to continue to multiply above the bottom two-thirds of the crypt and thereby to form dysplastic adenomas (7 , 30) . The particular mutations that underlie selection in culture are unknown. The differences in morphology of transformed foci (20 , 31) and the number of steps in transformation (32) indicate that many genetic loci are involved in the process. The effect of these mutations in culture is formally the same as that of APC in the colon in that both endow selective advantage to the cells under conditions that regulate the multiplication of normal cells. Rapid exponential multiplication of cultured cells minimizes or even selects against transformation (17) , perhaps because transformed cells tend to multiply, when unconstrained, at a lower rate than nontransformed cells (26, 27, 28) . The only parallel in vivo might be the relatively unconstrained cell growth of early embryogenesis during which tumor development is minimized.
Another form of selection for transformation in culture is to lower the concentration of growth factors while maintaining exponential growth at low population density (16 , 17) . The corollary in vivo would be a long-term reduction in the concentration of a hormone that stimulates cell multiplication. Accordingly it has been suggested that the diminution of testosterone production that occurs in aging men selects for testosterone-independent cells and contributes to the steep increase in prostate cancer beyond the age of 50 (33) . It has also been pointed out that deprivation of estrogenic hormones, which produces regression of spontaneous mammary tumors in mice, eventually results in progression of the residual neoplasia to a hormone-independent state (34) .
Selection also plays a role in chemical carcinogenesis of experimental animals. Farber has proposed the resistant hepatocyte model of liver carcinogenesis based on the findings that the cells in early stages of tumor development multiply in the presence of a carcinogen which is inhibitory to the growth of the normal hepatocytes (35) . There are a number of cases in rodents in which carcinogens apparently select for the growth of cells with preexisting mutations in ras genes (36 , 37) . Long-term repeated applications of nonmutagenic promoters alone give rise to a few liver tumors (38 , 39) which apparently result from selection of preexisting enzyme-altered cells that have a defect in growth control (39 , 40) . The compensatory hyperplasia that is an early step in experimental carcinogenesis of the skin (41) and colon (42) may also favor clonal expansion of cells that are defective in growth control and thereby promote the accumulation of mutations associated with progression.
If selection is to play the dominant role in carcinogenesis, there must, of course, be sufficient rogue clones from which to select. It was recently pointed out that conventional estimates of mutation frequencies in normal tissues may be far too low (43) . For example, 1 of 4,000 normal kidney cells in aging men was found to have a mutation at the X-linked HPRT locus (44) , which encodes a much smaller gene than many tumor suppressor genes (43) . Thus biallelic mutations of tumor suppressor genes may not be uncommon in normal tissues; mutations in micro- and minisatellites, some of which occur within functional genes, occur at much higher rates. There are enough cell divisions of normal intestinal stem cells and growing tumors to account for the 11,000 mutations/cell reported in colorectal polyps and carcinomas (45) without inferring an increased mutation rate.5 Simpson (43) suggests that the aging human body accumulates enough mutations to account for multistep carcinogenesis by selection of preexisting mutations. This would be especially true if selective conditions for clonal expansion were heightened with age. One indication that this is the case is that rat hepatocarcinoma cells are much more tumorigenic when inoculated into the liver of old rats than into that of young rats (46) . Another indication of tissue-wide changes with age is the slowdown in multiplication rates of intestinal mucosa accompanied by a large increase in heterogeneity among the crypt cells in the onset of DNA synthesis (47 , 48) .
In fact, the combination of accumulated mutations and disturbances of the regulatory environment associated with aging raises the question of why cancer is not more common than it is. It may be that the normal architecture of a tissue maintains a regulatory environment that keeps rogue clones behaving normally or disposes of them by apoptosis. This would be in keeping with the first principle of Elsasser’s theory of organisms, which postulates that "there can be regularity in the large where there is heterogeneity in the small: ‘order above heterogeneity’" (49) . This elementary principle has been expressed as macrodeterminism in developmental biology (50) and as holistic control of neuronal excitation in the brain (51) . A classical example is the teratocarcinoma of mice that is produced when young mouse embryos are grafted into the testes of adult mice (52) : inoculation of core cells of the teratocarcinomas into blastocysts results in their integration as normal elements in chimeric tissues of many types, including reproductively functional sperm (53) . The primary principle of ordered heterogeneity subsumes this behavior and several other genetic and epigenetic aspects of the cancer problem (54) . It therefore provides a theoretical foundation to account for the long-term stability of multicellular structures despite the accumulation of many somatic mutations (55, 56, 57) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from the
Council for Tobacco Research and the Elsasser Family Fund. 
2 Present address: Gene Logic, Inc., 2001 Center
Street, Berkeley, CA 94704.
3 To whom requests for reprints should be
addressed, at Department of Molecular and Cell Biology and Virus
Laboratory Life Sciences Addition, University of California, Berkeley,
CA 94720-3200. Phone: (510) 642-6617; Fax: (510) 643-6791; E-mail: hrubin{at}uclink4.berkeley.edu
4 The abbreviations used are: APC, adenomatous
polyposis coli; LDP, low density passage; MCDB 402, molecular,
cellular, and developmental biology medium 402; CS, calf serum; PD/D,
population doublings per day.
5 I. P. M. Tomlinson, personal communication.
Received 3/15/00. Accepted 9/12/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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H. Rubin Selected Cell and Selective Microenvironment in Neoplastic Development Cancer Res., February 1, 2001; 61(3): 799 - 807. [Abstract] [Full Text]
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, ++; 
, + and ++;
, ±, +, and ++. The degree of transformation of the uncloned A'
cultures for each serial assay is shown at the top of
each graph. The graph at the left is of the six serial
assays of the first set. The graph at the right is of
the four serial assays of the second set.