The level of genomlc instability was determined during tumor devel

[CANCER RESEARCH 54, 3173-3178,
June 15, 1994]
In Vivo Enhancement of Genomic Instability in Minisatellite Sequences of
Mouse C3H/10T½ Cells Transformed in Vitro by X-Rays'
and John
B. Little2
Harvard School of Publk Health, Laboratory ofRadiobiology,
The level of genomlc instability was determined during tumor devel
opment in vivo.Genomic rearrangements, a marker ofgenomlc instability,
was measured
mouse C3H/10T½ cells transfonned
in vitro by X-rays
with a DNA fingerprinting assay. Three transformed clones IsOlated from
type ifi foci were divided
into two groups
Cells from the first group
[email protected]. into syngeneic and noaimmunosuppressed C3H mice. After 3
to 5 months, the tumors were excised, and the neoplastic cells were
Isolated and subcloned. Cells from the second group were incubated in
vitro for 25 passages (about 6 months) to approximate the number of cell
divisions occurring in the tumor, and then they were subcloned. DNA was
from subclones grown in vitro and in vivo and analyzed with the
DNA fingerprinting assay. A high frequency of genomic rearrangements
(50-100%) was found in subclones derived from tumors that arose in vivo,
whereas the frequency
was very low (<10%) among subclones passaged in
be enhanced
present in the C3H mouse. In one clone (F-17) genomic Instability ap
peat-edto be activated and down regulated. The high frequency of insta
in tumor
cell subclones
did not appear
to result
an in
vivo selection of a more tumorigenic subpopulatlon of cells present In the
clone prior
to injection
in the animal.
This enhancement
genomic instability occurring in vivo could be required to complete the
process of transformation
to tumorigenicity
and allow the neoplastic
to adapt to a new environment.
It is now widely accepted that the development of cancer pro
ceeds by sequential steps from a normal cell to an invasive,
metastatic tumor (1—4).Many genes are involved in this process
including oncogenes, tumor suppressor genes, and genes involved
in angiogenesis and the development of metastases. Since new
oncogenes or tumor suppressor genes are still being discovered, it
is likely that we are now observing only a fraction of the relevant
mutations present in tumors (5). Nevertheless, the estimated num
ber of mutations necessary for tumor development can vary from as
many as 7 in the case of stomach cancer, up to 12 in the case of
prostate cancer (6). Estimations based on spontaneous mutation
rates in normal human cells cannot account for the large number of
mutations found in an individual tumor cell (5).
To explain the appearance of all these mutations in tumor cells,
genomic instability has been proposed as a important cellular process
in tumor heterogeneity and progression (2). The concept that genomic
instability can be an important process in human cancer also arises
from two other observations: (a) the incidence of most adult cancers
exhibits an exponential rate of increase with age, suggesting a series
of events each rendered more likely by the occurrence of a previous
event (7); (b) cancer predisposition can be a heritable event as
evidenced by the genetic chromosome instability syndromes, such as
Bloom's syndrome (8), as well as ataxia-telangiectasia patients whose
cells in vitro have an elevated frequency of translocations and a
10-fold increase in the loss of heterozygosity at the glycoprotein locus
(9). Ataxia-telangiectasia patients rarely live beyond 30 years of age,
and nearly 40% develop cancer (10).
Genomic instability includes all mechanisms that accelerate the
accumulation of damage or modifications to DNA, such as gene
amplification, translocation, recombination, or point mutation. Many
tumor cells exhibit rates of gene amplification, a type of genomic
instability, several orders of magnitude greater than their normal
counterparts (11, 12). Cells of high metastatic potential have been
reported to exhibit a 7-fold increase in mutation rate compared to cells
of low metastatic potential (13). The appearance of coincident muta
tions in TK6 lymphoblast clones exposed to X-rays (14) and the
acceleration of the spontaneous mutation rate at the hprt locus in
mutant Chinese hamster cells also supports the concept of genomic
instability (15).
Although these data clearly indicate the existence of genomic
instability, in vivo factors affecting its activation are not known, and
a causal relationship between genomic instability and tumor progres
sion remains to be established (16). The present study was thus
designed to examine whether the genomic instability could be stim
ulated by in vivo factors. The mouse fibroblast C3H/10T½ cell line
was used as model because in vitro transformation of these cells is
well documented (17—20),and their genomic instability can be mon
itored by measuring the genomic rearrangements occurring in a spe
cific minisatellite family with a DNA fingerprinting assay (21). The
frequency of genomic rearrangements in the minisatellite family
among clones growing as a tumor in syngeneic nonimmunosup
pressed C3H mice was compared with that in clones that underwent
approximately the same number of cell divisions in in vitro culture.
The relationship between the level of genomic instability and the
malignant potential of these transformed clones was also studied.
Cells and Culture
Mouse C3H embryo-derived
cells (10T½,clone 8) characterized by Reznikoff et aL (17) were grown in
Eagle's basal medium supplemented
with 10% heat-inactivated
Co.), penicillin
(50 units/mI),
fetal bovine
and streptomycin
Thesecells wereaneuploidwith a stablemodeof 81 chromosomesand
were used between passage 8—12.
Extraction of GenomicDNA DNAwas extractedaccordingto a salting
out procedure described by Miller et aL (22). Briefly, about 5 X i0@cells were
in phosphate-buffered
and centrifuged
a second
time. The cell pellet was resuspended in 3 ml of a buffer containing 10 mM
Tris-HCI, 400 mM sodium chloride, and 2 mp.i EDTA. To the cell suspension,
0.1 ml of SDS3 (20%) and 0.5 ml proteinase K (10 mg/rn]; DNase free) were
added and incubated overnight at 37°Cor at 50°Cfor 3 h. The DNA was
precipitated by the addition of 1.2 ml of 5 Msodium chloride. The tube was
by hand
1 mm and centrifuged
at 2500
for 15 mm;
supematant was transferred to another tube. The DNA was precipitated with
Received 2/3/94; accepted 4120/94.
charges. This article must therefore be hereby marked advertisement in accordance with
2.5 volumes of 95% ethanol, the tube was gently inverted for 30 s, and the
DNA was spooled out and air dried briefly. The DNA was dissolved in it
18U.S.C.Section1734solelyto indicatethisfact.
buffer (10 mM Tris-HCI-1 mM EDTA, pH 8.0), and RNase A (0.05 ml of a
Thecostsof publicationof thisarticleweredefrayedin partby thepaymentof page
1 This
a fellowshipto B. P. from“Lea
Fondade Ia Rechercheen Santedu Québec.―
2 To
at Harvard
3 The
Health, Laboratory of Radiobiology, 665 Huntington Avenue, Boston, MA 02115.
Downloaded from on April 23, 2015. © 1994 American Association for Cancer Research.
10 mg/mI solution) was added and incubated for 1 h at 37°C.The DNA was
a second
time with ethanol as described
above and redissolved
DNA Fingerprinting Analysis. Ten @gof each sample of DNA were
digested by Hinfi according to the recommendations of the manufacturer (New
England Biolabs, Inc., Beverly, MA). Digested DNA was separated in 0.8%
agarose gels (25 cm long). Electrophoresis was performed at 3.3 V/cm for 32
h; the gel was then soaked in 0.25 M HCI for 20 mm to induce chemical
cleavage of the DNA and then in 0.4 MNaOH for 40 mm. Transfer of DNA
-+ a1
onto Duralose-UV nitrocellulose membranes (Stratagene, La Jolla, CA) was
performed according to the procedure described previously (23). The mem
branes were baked for 2 h at 80°C;prehybridized for 3 h at 65°Cin 6 X SSC,
5 x Denhardt's, and 1% SDS; and hybridized for 16 h at 65°Cin 6 X SSC,
of 32P-labeledprobe M. The membranes were washed three times for 15 mm
at room temperature
in 1 X SSC-1% SDS and one to three times at 65°Cin
0.1% SSC-0.5% SDS. The washed filters were exposed at —70°C
to a Fuji RX
film with intensifying screens for 2 to 7 days.
The plasmid carrying the multilocus multiallele probe M used for this study
was provided by Dr. Brian J. Ledwith (Merck Sharp and Dohme Research
Laboratories, West Point, PA). Synthetically generated, probe M is based on a
minisatellite sequence found in the mouse major histocompatibility complex
(24) and is a tandem
of four nucleotides
(52 X 5'-AGGC).
This probe
was isolated from the plasmid as described previously (21).
Fig. 1. Banding pattem of DNA extracted from the livers of normal (host) mice that
was digested with Hinfi and analyzed on a DNA fingerprinting assay. Livers shown in
Tumorigenicity. Tumorigenicitytestingofcells isolatedfromType IIIfoci
and nontransformed C3H/10T½cells was carried out by inoculating 2 X 106
Lanes 3 and 4 were derived from host mice injected with the transformed clone shown
or 2 X l0@cells s.c. in the dorsal region of syngeneic nonimmunosuppressed
(X-ray-9 and F-17).
C3H mice (C3H/HeNcr1BR; Charles River). Four to nine animals were in
jected with each transformed cell clone. The number of progressively growing,
tumors that developed by 5 months after injection was scored.
DNA Fingerprinting Pattern from Normal C3H Mouse Cells.
The tumors used for this study were obtained after s.c. injection of
transformed 10T½cells in the back of C3H mice. The tumors were
not invasive, since they grow as a separate mass of tissue isolated
from the back muscles of the mice. Although some normal cells
required for the development of the tumors were present, such as
blood cells, the cell population in this type of tumor was clearly much
more homogeneous than for an invasive human tumor or an animal
tumor induced in vivo following exposure to a carcinogen. Neverthe
less, the cells isolated from the tumors were cultured in vitro for 6
weeks prior to extraction of DNA for the purpose of eliminating most
of the nontumor cells. In order to confirm that cells isolated from the
tumor and subcloned were the indeed derived from the 10T½trans
formed cells previously injected, their DNA fingerprinting pattern was
compared to the banding pattern of DNA isolated from the liver of the
host mouse. The DNA fingerprinting assay we used allows the detec
tion of minisatellite families characterized by a tandem repeat of 4 to
10 nucleotides dispersed throughout the genome of the cell. Since
each locus contains a different number of tandem repeats, digestion of
the genomic DNA with a restriction enzyme gives specific fragments
for each locus that can be easily visualized on an agarose gel. As these
loci are highly polymorphic, the banding pattern for these fragments
will vary from animal to animal.
DNA derived from the livers of mice carrying tumors derived from
the clones (X-ray-9 and F-17) as well as three additional mice was
analyzed. The livers were excised and trypsinized, and the DNA from
the single cell suspension was extracted as described in “Materialsand
Methods.―After digestion ofthe DNA with Hinfi, the banding pattern
of minisatellite sequences was obtained with the probe M. As is
shown in Fig. 1, none of these band patterns matched the ones
obtained with transformed 10T½cells (Figs. 2, 3, and 4). The DNA
fingerprinting pattern of liver from the mouse carrying the tumor
derived from clone X-ray-i I was not available. Thus, we cannot
exclude the possibility that a banding pattern might belong to the
normal host cells. Five new banding patterns were detected, which
obviously cannot be caused by a contamination of tumor by normal
mouse cells. Based on these data, we conclude that the modifications
detected in the subclones of cells isolated from the tumors have
occurred during development of the tumor and do not reflect contam
ination with host cells.
In Vivo Enhancement of Genomic Instability. This experiment
was undertaken to determine whether genomic instability in 10T½
cells transformed in vitro by X-rays can be enhancement factors
present in the host animal after injection in vivo. The three trans
formed clones analyzed were isolated from type III foci. The trans
formation of 10T½cells was performed in vitro with 6 Gy of X-rays
according to the established protocol (18). In a previous study (21),
genomic rearrangements in a family of minisatellite sequences hy
bridized with probe M was used as a marker of genomic instability
(21); 12 transformed clones were analyzed, five of which were tu
morigenic in syngeneic and nonimmunosuppressed C3H mice. Tu
morigenic clones with different levels of genomic instability were
chosen for the present study. These clones are X-ray-9 and X-ray-il,
which showed no detectable genomic instability, and clone F-17,
which was the most unstable with four genomic rearrangements
detected in cells isolated from the type III foci. As these rearrange
ments occurred in most of the cells as determined by Southern blots
(21), they presumably arose early in the process of transformation.
Cells from each of these three transformed clones were divided into
two groups. The first group was injected s.c. in syngeneic and non
immunosuppressed C3H mice. After 3 to 5 months, the tumors were
excised, and the tumor cells were isolated as described in “Materials
and Methods.―The tumor cells were then cultured for 6 weeks to
eliminate normal cells and subcloned into 96-well microtiter dishes.
As controls, DNA was extracted from the cells of the second group
immediately after their isolation from the type III foci. Additional
cells were cultured in vitro for 25 passages (i.e., 6 months or about
125 population doublings) to approximate the number of cell divisions
in the tumor
and then subcloned.
The DNA of these sub
clones isolated after 25 passages in vitro, as well as DNA from the
Downloaded from on April 23, 2015. © 1994 American Association for Cancer Research.
‘0 @, %O
9.4 kb .@
Fig. 2. DNA fingerprinting of subclone X-ray-9 derived from transformed cells propagated in vitro and in vivo.In vitro (Lanes on left) transformed cells and subclones derived from
tumor arising in vivo (Lanes on right). Lane 1, fingerprinting pattern of original clone X-ray-9.
subclones derived from the tumors that developed in vivo, was ex
tracted and analyzed with the DNA fingerprinting assay. This proce
dure allowed: (a) detection of genomic instability after a long period
of culture in vitro; and (b) determination of whether genomic insta
biity is stimulated by factors present in the animal.
Figs. 2, 3, and 4 illustrate the DNA fingerprinting analysis for the
subclones derived from transformed clones X-ray-9, X-ray-li, and
F-17, respectively. Following cultivation in vitro, no genomic rear
rangements were detected for the subclones from X-ray-9 (Fig. 2); the
same banding pattern was present in cells isolated immediately or
after 25 passages in vitro. One of nine subclones from X-ray-i 1 (Fig.
tion of genomic instability is not limited to a single pattern of genomic
In order to eliminate the possibility that these data might be the
result of in vivo selection of more transformed or more aggressive
subpopulations of cells present before injection into the animal, the
tumorigenic potential of randomly selected subclones derived from
tumors that developed in vivo was compared to that of their original
transformed clone. Since a subpopulation representing less than 1% of
the cells in original transformed clones can be present in the cell
population and not be detected with the DNA fingerprinting assay,
mice were injected with 2 X iO@cells of in vivo subclones instead of
3) andoneof eightfromF-17 (Fig. 4) showeda genomicrearrange 2 X 106 cells for the initial injection (Table 2). The tumorigenicity of
ment. In contrast to this low frequency of genomic rearrangements
each in vivo subclone was tested in four mice. After 5 months, no
following cultivation in vitro, new genomic rearrangements were
tumor appeared for four of the five in vivo subclones tested as well as
observed in 50—100%of the in vivo subclones studied (Table 1), and
for the original clones injected at this same low cell number. Only one
up to nine different banding patterns were detected (Fig. 2, 3, and 4).
mouse of four injected with 2 X iO@cells of the subclones X-ray-il
A-7 developed a tumor. These data indicate that the in vivo subclones
In vivo subclones from X-ray-li showed the highest degree of
genomic instability since five different banding patterns were ob
tested do not have a higher tumorigenic potential than the original
clones and suggest that the new pattern of genomic rearrangements
served. These data indicate that: (a) new genomics rearrangements
are not artifacts caused by an in vivo selection of a more aggressive
have occurred during tumor development in vivo; and (b) the activa
@S' )s.
Fig. 3. DNA fingerprinting of in vitro and in vivo subclones derived from original clone X-ray-i 1 (Lane 1).
Downloaded from on April 23, 2015. © 1994 American Association for Cancer Research.
Fig. 4. DNA fmgerprinting of in vitro and in vivo subclones derived from original clone F-17 (Lane 1).
and tumorigenic subpopulation of cells present in the original clone
before the injection in the animal.
Activity of Genomic Instability and Tumorigenic
Data are summarized in Table 1 regarding the frequencies of genomic
rearrangements in vitro and in vivo as well as the tumongenic poten
tial reported previously for each of the transformed clones studied. As
can be seen, clone X-ray-9 was the most stable after both in vitro and
in vivo culture and was also the most tumorigenic since all four mice
injected developed a tumor. On the other hand, clone F-i7 showed the
opposite behavior. Four genomic rearrangements were previously
detected for this transformed clone immediately after isolation (21),
and all in vivo subclones showed new genomic rearrangements. Nev
ertheless, only one mouse of eight injected with cells from original
clone F-i7 has developed a tumor (Table 1). Clone X-ray-il showed
an intermediate behavior.
Modulation of the Level of Genomic Instability. Our data also
indicate that the level of genomic instability can be activated and
down regulated. In a previous study, four genomic rearrangements
were detected early in the process of transformation for clone F-il. In
the present study, we report that a further in vitro culturing of clone
F-i7 for 6 months generated fewer genomic rearrangements. Indeed,
seven of the eight subclones derived from the original clone F-il
showed no detectable genomic instability. This finding is in contrast
to the level of genomic instability detected among subclones derived
from the tumor, thus after the growth of(culturing) clone F-il in vivo.
All subclones derived from this tumor showed new genomic rear
rangements. These data clearly indicate the possibility that the level of
genomic instability may be modulated, at least for clone F-il.
We have demonstrated that the level of genomic instability in
transformed C3H/iOT½ mouse cells can be modulated. These cells
were transformed in vitro by X-rays and genomic rearrangements, a
marker of genomic instability occurring after 25 subsequent passages
Frequencyof genomicrearrangements
among subclones―
in vitro or during the tumor development in the C3H mouse, were
by use of a DNA fingerprinting assay. This assay was used
to measure genomic rearrangements in a specific family of minisat
ellite sequences dispersed throughout the genome with a multilocus
and multiallele probe identified as M. With this protocol, it was
a Number
of in vitro
or in vivo
at least
one genomic
possible to measure the incidence of genomic rearrangements inde
number of subclones studied.
pendent of the direct action of the carcinogen on DNA.
b Number of animals with tumors/number of animals injected with 2 X i06 trans
It is interesting that very few new genomic rearrangements were
observed after 25 passages in vitro (or 6 months in culture). No event
was observed in X-ray-9 subclones, and only one subclone each from
Table 2 Malignant potential of 10T½transformed clones and their related
X-ray-il and F-il showed a genomic rearrangement. In contrast, we
in vivo subclones
have previously reported that a higher frequency of genomic rear
rangements occurs in the first six cell divisions following the second
event of the transformation process induced in vitro by X-rays, i.e., at
cells injected2X
the beginning of the formation of the type III foci (21). These data
suggest, at least for clone F-il, that genomic instability may be
initially activated by X-rays and down regulated during the process of
0/4X-ray-ilx-ray-i x-ray-9-A-74/40/4
transformation in vitro. Additional experiments are required to sup
port this observation.
x-ray-i i-A-7
The most important aspect of the present study is the observation
with a tumor/numberof animal injected.
that genomic instability in cells transformed previously in vitro can be
Table 1 Frequencies ofgenomic rearrangements among subclones derived from
transformed cells after growth in vitro and in vivo and tumorigenicity
initial transformed clone
of the
Downloaded from on April 23, 2015. © 1994 American Association for Cancer Research.
enhanced in vivo in the C3H mouse. After about the same duration of
time and number of cell divisions as for the in vitro culture, new
genomic rearrangements were observed in all the three clones tested,
even in those found to be previously stable in vitro. Since the cells
were previously exposed to X-rays in vitro, these new genomic
rearrangements were induced in vivo after exposure to the carcinogen.
Enhancement of the frequency of gene amplification and point
mutations by toxic agents, two types of genomic instability, has
already been reported for malignant cells in vitro. When incubated
with N-(phosphonoacetyl)-L-asparate,
highly tumorigenic cell lines
amplified the genes that code for a multifunctional enzyme complex
that contains carbamyl phosphate synthase, aspartate transcarbamy
lase, and dihydro-orotase (CAD) activities, at a frequency of iO@,
whereas in normal counterparts the event is undetectable by the
method used in these studies (ii, 12). However, it was still not
demonstrated that such enhancement of the activity of genomic insta
biity can also occur in the animal and that this cellular process could
play a role in tumor development (25).
Why can genomic instability be enhanced in vivo in 10T½cells
transformed in vitro by X-rays? One possible interpretation is that
transformation was incomplete in vitro and additional genetic modi
fications were required to obtain a fully tumorigenic cell. In this
regard, the growth of weakly immunogenic tumors may actually be
stimulated by an immune reaction (26). Such a process might be
involved in the in vivo enhancement of genomic instability reported in
this study, giving rise to a growth advantage that facilitates the
development of a tumor.
On the other hand, genomic instability can be an important part of
a mechanism allowing the transformed cell to adapt to a new envi
ronment. The cells that produced the tumor may have the ability to
continue on with the genomic rearrangements, resulting in some
malignant cells ultimately carrying the most favorable properties for
their progression to metastatic cells and to evade host nonimmune and
immune responses (21—34).In this regard, the capacity to modulate
genomic instability could also be very important for the survival of the
tumorigenic cell. An uncontrolled expression of genomic instability
could be responsible for the accumulation of too many DNA modi
fications that would eventually prove to be lethal such that the cell
would die before forming a tumor, or conversely, that tumor regres
sion might occur. This latter hypothesis can, at least in part, be
supported by our results showing that clone F-il demonstrated the
highest level of genomic rearrangement in vitro and in vivo but a
lower tumorigenic potential. However, more research is still required
to confirm this hypothesis.
Another interesting aspect of genomic instability that we observed
with our DNA fingerprinting assay is its specificity of action. In a
previous report, four multilocus and multiallele probes detecting dif
ferent minisatellite families dispersed through the genome were used
to detect the genomic rearrangements induced in cells transformed by
X-rays (21). Only the minisatellite family detected with the probe
identified as M showed genomic rearrangements. This specificity
cannot be attributed to direct action of the carcinogen, since X-rays
interact essentially randomly within the genome. What cellular pro
cess can account for the observations that genomic instability occurs
in specific regions of the genome, and can be modulated during
and tumor development? Multiple genes show
genomic stability functions, such as those controlling accurate dupli
cation and distribution of DNA in progeny cells and the fidelity of
repair (i6, 35). Recently, a mutation in genes controlling the cell cycle
(36—37), such
as the suppressor
gene p.53, was correlated
age, they can only in part account for the modulation and accumula
tion of mutations in a specific region of the genome.
In conclusion, genomic instability appeared to be a early event in
our cell system, occurring during the proliferation of cells surviving
X-ray exposure; subsequently, the activity of this mechanism may be
modulated to allow the cell to complete its transformation in vivo or
to grow in this new environment.
We thank Dr. Brian T. Ledwith for providing probe M.
I. Foulds, L. The experimental study of tumor progression: a review. Cancer Res., 14:
2. Nowell, P. C. The clonal evolution of tumor cell populations. Science (Washington
DC), 194: 23-28, 1976.
3. Pitot, H. C., Ooldsworthy, T., and Moran, S. The natural history of carcinogenesis:
implications of experimental carcinogenesis in the genesis of human cancer.
J. Supramol. Struct. Cell. Biochem., 17: 133—146,
4. Weinstein, I. B. The origins of human cancer: molecular mechanisms of carcinogen
esis and their implications for cancer prevention and treatment. Cancer Res., 48:
4135—4143, 1988.
5. Loeb, L. A. Mutator phenotype may be required for multistage carcinogenesis. Cancer
Res., 51: 3075—3079,1991.
6. Renan, M. J. How many mutations are required for tumorigenesis? Implications from
human cancer data. Mol. Carcinog., 7: 139—146,1993.
7. Armitage,
P., and Doll, R. The age distribution
of cancer and a multi-stage
theory of
carcinogenesis. Br. J. Cancer, 8: 1—12,
8. German,
J. Patterns
of neoplasia
the chromosome-breakage
dromes. in: 1. German (ed), Chromosome Mutation and Neoplasia, pp. 97—134.
York: Alan R. Liss, 1983.
9. Bigbee, W. L., Langlois, R. G., Swift, M., and Jensen, R. H. Evidence for an elevated
frequency of in vivo somatic cell mutations in ataxia-telangiectasia. Am. J. Hum.
Genet., 44: 402—408,1989.
10. Peterson, R. D. A., Funkhouser, J. D., Tuck-Muller, C. M., and Gatti, R. A. Cancer
susceptibility in ataxia-telangiectasia. Leukemia (Baltimore), 6 (Suppl. I): 8—13,
11. Tlsty, T. D., Margolin, B. H., and Lum, K. Differences in the rates of gene amplifi
cation in nontumorigenic and tumorigenic cell lines as measured by Luria-Delbruck
fluctuation. Proc. NatI. Acad. Sd. USA, 89: 9441—9445,1989.
12. Wright, J. A., Smith, H. S., Watt, F. M., Hancock, M. C., Hudson, D. L, and Stark,
0. R. DNA amplification is rare in normal human cells. Proc. NatI. Acad. Sci. USA,
87: 1791—1795,1990.
13. Cifone, M. A., and Fidler, I. J. Increasing metastatic potential is associated with
increasing genetic instability of clones isolated from murine neoplasms. Proc. NatI.
Acad. Sci. USA, 78: 6949—6952,1981.
14. Li,C-Y.,Yandell,D.W.,andLittle,J. B.Evidenceforcoincidentmutationsinhuman
lymphoblast clones selected for functional loss of a thymidine kinase gene. Mol.
Carcinog., 5: 270—277,1992.
15. Chang, W'. P., and Little, 1. B. Persistently
elevated frequency
of spontaneous
mutations in progeny of CHO clones surviving X-irradiation: association with de
layed reproductive death phenotype. Mutat. Res., 270: 191—199,
16. Cheng, C. C., and Lawrence, A. L. Genomic instability and tumor progression:
mechanistic considerations. Adv. Cancer Res., 60: 121—156,
17. Reznikoff, C. A., Bertram, 1. S., Brandow, D. W., and Heidelberger, C. Quantitative
and qualitative studies of chemical transformation of cloned C3H mouse embryo cells
sensitive to postconfluence inhibition of cell division. Cancer Rca., 33: 3239—3249,
18. Terzaghi,M., and Little,J. B. X-radiation-induced
transformationin a C3Hmouse
embryo derived cell line. Cancer Rca., 36: 1367—1374,
19. Miller, R. C., Geard, C., Geard, M. J., and Hall, E. J. Cell cycle-dependent radiation
induced oncogenic transformation of C3H/lOT½cells. Radiation Res., 130:
20. Leuthauser, S. W. C., Thomas J. E., and Guernsey,
D. L. Oncogenes
in X-ray
transformed C3H/10T½mouse cells and in X-ray-induced mouse fibrosarcoma
(RIF-1) cells. tnt. J. Radial. Biol., 62: 45—51,1992.
21. Paquette, B., and Little, J. B. Genomic rearrangements
in mouse C3H/10T½ cells
transformed by X-rays, UV-C, and 3-methylcholanthrene detected by a DNA finger
print assay. Cancer Res., 52: 5788—5793,1992.
22. Miller, S. A., Dykes, D. D., and Polesky, H. F. A simple salting out procedure for
extracting DNA from human nucleased cells. Nucleic Acid Res., 16: 1215, 1988.
23. Ledwith, B. 1., Storer, R. D., Prahalada, S., Manam, S., Leander, K. R., van Zwieten,
M. J., Nichols, W. W., and Bradley, M. 0. DNA fingerprinting of 7,12-dimethylben
and spontaneous CD-I mouse liver tumors. Cancer Res., 50:
Kobari, J. A., Strauss,
E., Minard, K., and Hood, L. Molecular
of the hotspot
of recombination in the murine major histocompatibility complex. Science (Wash
with the
appearance of gene amplification. Although a mutation in one of these
cell functions could increase the overall accumulation of DNA dam
ington DC), 234: 173—179,1986.
25. Hartwell, L. Defects in a cell cycle checkpoint may be responsible for the genomic
instability of cancer cells. Cell, 71: 543—546,1992.
Downloaded from on April 23, 2015. © 1994 American Association for Cancer Research.
26. Prehn, R. L Stimulatory effects of immune reactions upon the growths of untrans
33. Nicolson, 0. L, and Poste, G. Tumor implantation and invasion at metastatic sites.
planted tumors. Cancer Res., 54: 908—914,1994.
27. Foulds, L. Neoplastic development.
Int. Rev. Hap. Pathol., 25: 77—181,1983.
New York: Academic Press, 1975.
34. Nicolson, 0. L Tumor progression,
28. Medina,D. Tumorprogression.In: F. F. Becker(ed), Cancer:A Comprehensive
Treatise, Vol. 3, pp. 99—119.New York: Plenum Publishing Corp., 1976.
29. Nowell, P. C. Mechanisms
of tumor progression.
and the evolution of metastatic
phenotype diversity. Qin. Exp. Metastasis, 2: 85-105, 1984.
35. Fishel, R., l@escoe,M. K., Rao, M. R. S., Copeland, N. G., Jenkins, N. A., Gather, J.,
Cancer Res., 46: 2203-2207,
30. Nicolson, G. L Generation of phenotypic diversity and progression in metastatic
tumors.CancerMetastasisRev.,3: 25-42, 1984.
31. Filder,I. J., andHart,I. R. Biologicaldiversityin metastaticneoplasms,originsand
implications. Science (Washington DC), 217: 998—1003,1982.
Kane, M., and Kolodner, R. The human mutator gene homolog MSH2 and its
association with hereditary nonpolyosis colon cancer. Cell, 75: 1027—1038,
36. Livingstone, L R., White, A., Sprouse, J., Livanos, E., Jacks, T., and Thty, T. D.
Altered cell cycle arrest and gene amplification potential accompany loss of wild-type
p53. Cell, 70: 923—936,1992.
37. Yin, Y., Tainsky, M. A., Bischoff, F. Z., Strong, L, and Wahi, G. M. Wild-type p53
32. Owens, A. H., Coffey, D. S., and Baylin, S. B. (eds.). Tumor cell heterogeneity: origin
and implications. New York: Academic Press, 1982.
restores cell cycle control and inhibits gene amplification in cells with mutant p53
alleles.Cell,70: 937—948,
Downloaded from on April 23, 2015. © 1994 American Association for Cancer Research.
In Vivo Enhancement of Genomic Instability in Minisatellite
Sequences of Mouse C3H/10T½ Cells Transformed in Vitro by
Benoit Paquette and John B. Little
Cancer Res 1994;54:3173-3178.
Updated version
E-mail alerts
Reprints and
Access the most recent version of this article at:
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from on April 23, 2015. © 1994 American Association for Cancer Research.