[CANCER RESEARCH 41, 3024-3029,
0008-5472/81/0041-OOOOS02.00
August 1981]
Restriction Endonuclease Studies of Hyperplastic Outgrowth Lines from
BALB/cfCSH Mouse Hyperplastic Mammary Nodules1
Robert D. Cardiff,2 Thomas G. Fanning, David W. Morris, Rhoda L. Ashley,3 and Leslie J. Faulkin
Department of Pathology, School of Medicine ¡R.D. C., T. G. F., D. W. M., R. L. A.], and Department
University of California, Davis, California 956)6
ABSTRACT
The DNA's isolated from five mouse hyperplastic
mammary
gland outgrowth lines from BALB/cfC3H mice were digested
with the restriction endonucleases Psil, ßamHI,or EcoRI; electrophoresed; and analyzed by Southern blotting and autoradiography. Proviral DMA sequences from the acquired C3H
mouse mammary tumor virus were detected in the DNA of all
five lines, indicating that they were infected. The DNA of the
five hyperplastic lines contained more EcoRI and ßamHImouse
mammary tumor virus proviral DNA fragments than did DNA
from normal organs, suggesting that the hyperplastic tissues
were composed of more homogeneous cell populations than
was lactating mammary gland. Each hyperplastic line had
unique and reproducible ßamHIand EcoRI restriction (integra
tion) patterns which were stable over as many as seven trans
plant generations. Three sublines, which originated from the
same hyperplastic alveolar nodule, had unique integration pat
terns but also shared several fragments. On the basis of these
observations, we propose that mouse mammary "hyperplasias" are clonal dominant premalignant neoplasms.
INTRODUCTION
Restriction endonuclease
mapping of MuMTV4 proviral DNA
has provided important data concerning the origin of various
proviruses (5), the acquisition of exogenous virus (4, 8), and
the cellular composition of mammary tumors (3, 4, 6-9). Re
striction endonuclease mapping has been particularly useful in
the study of MuMTV proviral DNA in BALB/cfC3H mammary
tumors and in normal mammary gland (4). We now report the
application of this technique to the study of premalignant
hyperplastic mammary gland outgrowths from BALB/cfC3H.
The hyperplastic outgrowths were developed from BALB/
cfC3H HAN's which were serially transplanted in BALB/c
recipients (1). We developed and characterized 5 outgrowth
lines from 3 HAN's in order to study the virology and biology of
this high-risk mammary tissue. These outgrowths, referred to
as the Z lines, are biologically stable, morphologically benign
mammary tissues which do not invade surrounding tissue and
do not metastasize. However, they do develop mammary tu
mors more frequently than normal ducts (2). Therefore, the Z
' Supported by USPHS Contract NO1-CP-61013
from the Virus Cancer Pro
gram. Division of Cancer Cause and Prevention. National Cancer Institute and by
USPHS Grant 5R01-CA 21454 from the National Cancer Institute.
2 To whom requests of reprints should be addressed.
3 Present address: Department of Microbiology and Immunology. School of
Medicine, University of Washington, Seattle, Wash. 98195.
4 The abbreviations used are: MuMTV, mouse mammary tumor virus; HAN,
hyperplastic alveolar nodule.
Received December 1. 1980: accepted April 22, 1981.
3024
ol Anatomy,
School of Veterinary Medicine [L. J. F.],
lines fit the operational definition of mammary preneoplasia (1,
2).
We have documented previously that each outgrowth line is
morphologically and biologically unique (1). Differences in the
tumor incidences of the outgrowth lines could not be correlated
with growth rate, subgross morphology, or histology (1 ). Tumor
incidences could not be correlated with quantity of MuMTV
RNA per cell or the proportion of cells containing MuMTV
antigens (1).
The studies reported here extend our virological studies of
the Z lines to the DNA level. We present evidence, based on
restriction endonuclease mapping, that all of the Z lines contain
acquired C3H MuMTV proviral DNA and each has unique EcoRI
and ßamHIrestriction map patterns which are stable over many
transplant generations. These results have important implica
tions concerning the nature of mouse mammary preneoplasia.
MATERIALS
AND METHODS
Outgrowths and Animals. The hyperplastic outgrowths were
developed from BALB/cfC3H HAN's and maintained by serial
transplantation into gland-free mammary fat pads as described
previously (1). The host mice were 3-week-old female BALB/
c mice obtained from Simonsen Laboratories, Gilroy, Calif.
Following transplantation (1), the mice were maintained as
virgins, 5/cage, under controlled conditions of temperature
and light. Food and water were given ad libitum. Animals were
maintained under the guidelines set forth by the University of
California.
Nucleic Acid Extraction. Individual mammary fat pads, con
taining a single transplant, were minced and then disrupted by
homogenizing with a Dounce homogenizer in TEN buffer [10
rriM Tris-HCI (pH 7.4), 10 rnw EDTA, and 10 mw NaCI; 4, 7].
Proteinase K and sodium dodecyl sulfate were added to the
homogenate to give final concentrations of 100 to 250 jug/ml
and 0.5%, respectively. The mixture was incubated 30 to 60
min at 37°and then extracted twice with phenol. The extracted
nucleic acids were dialyzed exhaustively at 4°against 10 mw
Tris-HCI (pH 8.0) and 1 mw EDTA to remove the residual
phenol and stored at 4°.
MuMTV virions (from the Mm5mt/c1 cell line supplied by the
Frederick Cancer Research Center, through the Research Re
sources, Biological Carcinogenesis Branch, National Cancer
Institute) were disrupted by the addition of sodium dodecyl
sulfate to 1%, and the RNA was extracted twice with phenol.
The MuMTV RNA was heated to 90°for 3 min and centrifuged
in a 5 to 20% sucrose gradient (11 ). The material sedimenting
in the 18 to 28S region of the gradient was collected, ethanol
precipitated and dissolved in a small volume of 10 rriM Tris-HCI
(pH 7.4), 50 m.M KCI, and 1 mW EDTA; and the material was
stored at -20°.
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Restriction
Maps of Hyperplastic
Outgrowths
Preparation of MuMTV 12P-radiolabeled Complementary
DMA. MuMTV-specific 32P-radiolabeled complementary DMA
was prepared as described previously using MuMTV RNA, calf
thymus primers, radiolabeled dATP, and avian myeloblastosis
virus reverse transcriptase (provided by Dr. J. Beard, Life
Sciences, Inc., St. Petersburg, Fla., through the Research
Resources, Biological Carcinogenesis Branch, National Cancer
Institute) (4, 7, 8).
Enzyme Digests and Blotting. Restriction enzymes were
purchased from New England Biolabs. Beverly, Mass. Digests
were done in 100 rriM Tris-HCI (pH 7.4), 10 mM NaCI, 5 mw
MgCI2, and 100 fig lysozyme per ml at 37°(8). Cellular DNA
15.6 9m
6.4 —
4.4
2.9 —
2.5
(5 to 10 jug) was mixed with ~1 fig of bacteriophage \ DNA
before enzyme digestion. The digested DNA's were electrophoresed in 1% agarose gels. The A restriction pattern visual
ized under UV after ethidium bromide staining was used to
verify the completeness of digestion. The transfer of DNA
fragments to nitrocellulose filters was done as described by
Southern (13). After transfer, the filters were treated, washed,
and autoradiographed according to published procedures (4).
1.5
1.3
0.6
RESULTS
Origin and Current Status of the Z Hyperplastic Outgrowth
Lines. The 5 hyperplastic outgrowth lines, designated the Z
lines, were developed from three BALB/cfC3H HAN's (1). The
Z5 sublines came from the same HAN. Z5d and Z5c came from
different second generation transplants. Twenty days after the
original Z5c transplantation, the donor of Z5c was reopened
and fragments of the same outgrowth were transplanted into
additional mice. This second series of transplants was desig
nated Z5ci. The Z3 and Z4 lines were developed from separate
HAN's.
We reported previously on the first 6 transplant generations
of the outgrowth lines (1). The lines are currently in transplant
generation 13. They have retained the biological and morpho
logical characteristics reported previously. The data reported
in this paper came from transplant generations 4 and 8 to 11.
Acquired C3H Proviral DNA. The enzyme Psfl cleaves the
milk-transmitted acquired C3H MuMTV proviral DNA at 5 sites
resulting in a characteristic series of internal DNA fragments
(4). In a like manner, ßamHIcleaves C3H MuMTV proviral DNA
twice, generating a single internal fragment (4). Since cleavage
of the endogenous BALB/c MuMTV provirus DNA by these
enzymes results in a different pattern, the acquired C3H
MuMTV proviral DNA can be readily identified in autoradiographs of Psfl- or ßamHI-cleaved BALB/c DNA which has
1
3
4
8
Fig. 1. Psfl restriction pattern of MuMTV proviral sequences in the 5 BALE/
cfC3H hyperplastic outgrowth lines and in spleen DNA's. Each DNA (5 to 10 fig)
was digested with PsH and analyzed by the Southern blotting procedure and
autoradiography. Slot 1, 32P-A DNA digested with H/ndlll to yield fragments of
15.6, 6.4, 4.4, 2.9, 1.5, and 1.3 x 106 daltons; Stof 2 and 8. BALB/cfC3H
spleen showing the endogenous proviral MuMTV Psfl restriction pattern of BALB/
c; Slot 3, 23 generation 8; Slot 4, Z4 generation 8; Slot 5, Z5c generation 8; Slot
6, Z5c> generation 8; Slot 7, Z5d generation 8. The numbers 2.5 and 0 6 indicate
the location of the 2.5 and 0.6 x 10" dalton fragments from the acquired C3H
MuMTV proviral DNA.
acquired C3H MuMTV DNA are created with these enzymes.
The size of each fragment is dependent upon the length of the
host sequences. When a significant fraction of the cells in a
given population contain a C3H MuMTV provirus integrated
into the same host site, it is detectable in EcoRI or ßamHI
restriction endonuclease maps as discrete bands (16). The
pattern of discrete bands observed in autoradiographs is re
ferred to here as the integration pattern.
Autoradiographs of the 5 Z line DNA's following digestion
with ßamHIor EcoRI revealed that each line had more detect
able MuMTV fragments than did normal organ DNA (Figs. 2 to
6). These additional fragments are attributable to the acquired
C3H MuMTV proviral DNA. Each outgrowth line had a unique
pattern of MuMTV DNA fragments. For example, Z3 had a
characteristic triplet (5.6, 4.7, and 4.4 x 106 daltons) of
been infected with C3H MuMTV (4).
The DNA from all of the outgrowths from the Z lines contained
the 2.5 and 0.6 X 106 dalton Psrl MuMTV fragments (Fig. 1)
and the 0.65 x 106 dalton ßamHIMuMTV fragment character
MuMTV ßamHIDNA fragments which were not found in any
other line (Fig. 2). Z4 had a number of C3H MuMTV BamHI
fragments but also had a characteristic triplet (5.4, 4.5, and
4.2 X 106 daltons) not found in the other lines (Fig. 2).
istic of the acquired C3H provirus (Fig. 2). These fragments
are direct evidence that the outgrowths contain C3H MuMTV
proviral DNA.
Integration Patterns. The restriction enzyme EcoRI cleaves
the acquired C3H MuMTV proviral DNA at a single site near
the center of the genome (4, 5). ßamHI cleaves the C3H
MuMTV proviral DNA twice near the center of the genome (4,
16). Both enzymes also cleave at variable distances into the
host flanking sequences. Thus, 2 large fragments for each
Since the 3 Z5 sublines were developed from a single HAN,
a comparison of their integration patterns was of special inter
est. Each line had a unique set of "marker" C3H MuMTV
proviral DNA fragments. For example, Z5c has a 6.5 x 106
dalton ßamHIfragment, while Z5c, had a 6.8 x 106 dalton
ßamHI fragment, and Z5d had a 8.4 x 106 dalton ßamHI
fragment, none of which were found in the other Z lines (Fig.
2).
The Z5 sublines also appeared to have C3H MuMTV DNA
AUGUST 1981
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3025
R. D. Cardiff
et al.
|15.6
6.4
4.4
2.9
1.5
1.3
0.65
l
8
Fig. 2. BamHI restriction pattern of MuMTV proviral sequences in the 5
BALB/cfCSH hyperplastic outgrowth lines and in spleen DNA's. Each DNA (5 to
10 /ig) was digested with SamHI and analyzed by Southern blotting and autoradiography. Slot 1, 23 generation 9; band markers at left of column indicate the
distinctive 5.6, 4.7, and 4.4 x 106 dalton 23 fragments. Slot 2. 24 generation
11; markers at left indicate the distinctive 5.4, 4.5. and 4.2 x 106 dalton 24
fragments. Slot 3, Z5c generation 8; marker at left indicates the distinctive 6.5
x 106 dalton Z5c fragment. S/of 4, Z5ci generation 8; marker at left indicates
the distinctive 6.8 x 106 dalton Z5c, fragment. Slot 5, Z5d generation 8; marker
at left indicates the distinctive 8.4 x 10e dalton Z5d fragment. Slot 6. an equal
mixture of Z5c, ZSCi, and Z5d DNA's; markers at left indicate the shared 10.2,
5.2, 4.4, and 1.7 x 106 dalton fragments. Slot 7, BALB/cfC3H spleen DMA
showing the SamHI pattern of the endogenous MuMTV proviral DNA's. S/ot 6,
32P-X DMA digested with Hind\\\. The number 0.65 indicates the location of the
0.65 x 10" dalton SamHI fragment of the acquired C3H MuMTV proviral DNA.
Fig. 3. EcoRI restriction
patterns of MuMTV proviral DNA's comparing three
individual Z5d hyperplastic outgrowths from generation 8. Each outgrowth DNA
(5 to 10 fig) was digested with EcoRI and analyzed by Southern blotting and
autoradiography. S/of Õ,BALB/cfC3H spleen DNA showing the EcoRI pattern of
the endogenous MuMTV proviral DNA; S/ofs 2, 3, and 4, Z5d generation 8.
fragments of the same size. For verification, the DNA from the
Z5 sublines was mixed, digested with SamHI, and mapped
(Fig. 2). The DNA mixture had more detectable fragments than
did the spleen DNA. Fragments at 10.2, 5.2, 4.4, and 1.7 x
106 daltons could be observed in the DNA of 2 or more of the
lines and in the DNA mixture. The 5.2 and 1.7 x 106 dalton
bands appeared in all 4 Z5 samples. On the other hand, the
10.2 x 106 dalton fragment did not appear in Z5d, while the
4.4 x 106 dalton fragment did not appear in Z5ci.
Integration Pattern Stability. In order to assess the reproducibility of the integration patterns, the DNA of 3 samples of
generation 8 transplants of Z5d and 5 samples of generation
4 transplants of Z4 were digested with EcoRI, electrophoresed,
and analyzed by Southern blotting and autoradiography. The
autoradiographs of the 3 Z5d transplant DNA's were identical
(Fig. 3). The 5 samples of Z4 transplant DNA's and a pool of 4
other Z4 generation 4 transplant DNA's were also identical
(Fig. 4). Thus, all transplant DNA's from the same generation
had the same integration patterns.
Next, DNA's from a number of different transplant genera
tions were compared for possible variation following EcoRI and
SamHI digestion. The autoradiographs of generations 8 and 9
from Z3 were identical (Fig. 5). Similarly, the Z4 DNA from
generations 4, 8, and 11 contained no detectable differences
in integration patterns (Fig. 5). The autoradiographs of gener-
3026
I
3
4
Fig. 4. EcoRI restriction patterns of MuMTV proviral DNA comparing individual
Z4 outgrowths from generation 4. All DNA's (5 to 10 /ig) were digested with
EcoRI and analyzed by Southern blotting and autoradiography. Slot 1, BALB/
cfC3H spleen DNA showing the pattern of endogenous MuMTV proviral DNA;
Slots 2, 3, 5, 6 and 7, Z4 outgrowth; Stof 4, a pool of 4 Z4 outgrowths from
generation 4.
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Restriction
Maps of Hyperplastic
Outgrowths
ations 8 and 11 of the Z5 series DNA's were also characteristic
of each subline (Fig. 6). The corresponding EcoRI digestions
also resulted in identical maps (data not shown). Thus, the C3H
MuMTV proviral integration patterns were stable over the gen
erations studied.
15.6
6.4
4.4
—
.
DISCUSSION
: : —
-'
2.9
1.5
1.3
i
8
Fig. 5. SamHI restriction patterns of MuMTV proviral DNA comparing three
transplant generations of Z4 and 2 transplant generations of Z3. The outgrowth
DNA's (5 to 10 jug) were digested with SamHI and analyzed by Southern blotting
and autoradiography. Slot 1. 32P-A DNA digested with Hmdlll; Slot 8, BALB/
cfC3H spleen DNA showing the BamHI pattern of endogenous MuMTV proviral
DNA; Slots 2, 3, and 4, Z4 outgrowth from generations 4, 8, and 11, respectively.
Slots 5, 6, and 7, Z3 outgrowth from generations 8, 9, and 9, respectively.
15.6
6.4
-EH
4.4
2.9
to
IH
ME r
1.5
1.3
i
Fig. 6. Bam HI restriction patterns of MuMTV proviral DNA comparing 2
transplant generations of Z5c, Z5c,, and Z5d. The outgrowth DNA's (5 to 10 /jg)
were digested with SamHI and analyzed by Southern blotting and autoradiogra
phy. S/ot T, Hmolll-digested
32P-lambda DNA; Slot 8, BALB/cfC3H
spleen
showing the SamHI pattern of endogenous MuMTV proviral DNA; S/ofs 2 and 3,
Z5c outgrowth from generations 8 and 11, respectively; Slots 4 and 5, Z5c,
outgrowth from generations 8 and 11, respectively; Slots 6 and 7. Z5d outgrowth
from generations 8 and 11, respectively.
AUGUST
Certain mammary hyperplasias of mice and humans are
considered preneoplastic because they have a high risk of
developing into malignant tumors (2, 10, 17). Since multiple
transplants taken from a single mouse hyperplastic lesion may
exhibit different morphological and biological characteristics,
each mammary hyperplasia is thought to be composed of a
heterogeneous cell population with varying biological potentials
(2, 3, 9, 10). However, the concept of heterogeneity of cells in
breast hyperplasia has not been rigorously tested.
Recently, studies of restriction maps of MuMTV DNA from
BALB/cfC3H and GR/A mouse mammary tumors have sug
gested that each tumor arises as a distinct subset of cells from
a heterogeneous mammary epithelial cell population (4, 7, 16).
Mammary tumors, however, rarely arise directly from normal
mammary epithelium but generally arise from the precursor
lesion, mammary hyperplasia (2, 10). The experiments re
ported here with the Z line hyperplastic outgrowths represent
the first study of the premalignant breast tissue with the restric
tion endonuclease and Southern transfer techniques.
The rationale for using restriction endonuclease maps as
evidence of homogeneity of BALB/cfC3H tumors is based on
the use of enzymes, such as EcoRI and SamHI which cleave
near the center of the MuMTV proviral DNA and in the flanking
host DNA, and on the comparison of the EcoRI and SamHI
maps of tumor and lactating mammary gland DNA's (4). The
basis for interpretation of this procedure has been described
in detail elsewhere (16).
Briefly, infection of BALB/c mammary cells with the milktransmitted C3H MuMTV results in the integration of C3H
MuMTV proviral DNA into many sites in the host DNA, with
different sites being used randomly in different cells (4, 16).
Since the progeny of a single cell does not become a major
portion of the infected but nonneoplastic mammary population,
the acquired C3H MuMTV proviral DNA's are not detected as
discrete EcoRI fragments in restriction maps. Thus, when DNA
from infected BALB/c tissues lack discrete EcoRI C3H MuMTV
proviral DNA fragments, the tissues are random or heteroge
neous with respect to viral integration sites. BALB/cfC3H
lactating mammary gland DNA does not contain detectable
EcoRI C3H MuMTV proviral DNA fragments and can be con
sidered a random population (4).
If the progeny of one infected BALB/c mammary cell gains
a selective advantage as a tumor, they may become the major
cell type in the population tested. The DNA from the population
should then contain one predominant set of C3H MuMTV
proviral DNA fragments. If all or most cells contain the same
set, the C3H MuMTV DNA would be detectable as discrete
bands following EcoRI or SamHI digestion. On the basis of this
interpretation, GR/A, C3Hf, and BALB/cfC3H
tumors are
thought to be composed primarily of cells derived from one or
a few cells (4, 7,16). This implies a clonal or quasiclonal origin
of these tumors (3, 6, 7, 16).
Acquired proviral DNA's have also been detected in murine
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3027
R. D. Cardiff
et al.
and avian leukemia systems using enzymes which cleave the
provirai DNA once (12, 14, 15). Restriction maps of these
DNA's have also been considered as reasonable evidence of
the clonal origin of tumors in these tumor systems associated
with RNA tumor virus (12, 15). The clonal origin of tumors is a
widely accepted concept (3).
Restriction mapping, however, cannot distinguish between a
population composed of a single clone and a population com
posed of several clones or a population composed of a mixture
of detectable and nondetectable clones. As a result, it is difficult
to assert that, on the basis of restriction mapping alone, mouse
mammary tumors are monoclonal. The presence of detectable
proviral restriction fragments does require that a significant
fraction of cells contain the same proviral fragment. In this
sense, the mammary tumors are at least composed of nonrandom clonal dominant populations. We will also refer to them as
homogeneous as compared to the heterogeneous (random)
mammary epithelial cell populations (4, 16).
The development of the hyperplastic outgrowth Z lines pro
vided the opportunity to determine if premalignant tissue re
sembles malignant or normal mammary tissue. The presence
of Psrl and SamHI fragments characteristic of the acquired
C3H proviral DNA provided direct evidence that all of the Z
lines were infected. The presence of additional C3H MuMTV
proviral DNA fragments in each line provided evidence that the
hyperplastic mammary outgrowth lines had amplification of
MuMTV genes and were a nonrandom cell population. In this
respect, the outgrowth lines more closely resemble malignant
than normal mammary epithelium (4, 6-9, 16). This is, to the
best of our knowledge, the first evidence of a homogeneous
population in hyperplastic breast tissue of any type and con
flicts with the general concept of tissue heterogeneity refer
enced previously.
It is possible that the homogeneity of these outgrowths is a
result of the selective pressure inherent in serial transplanta
tion. While this is a plausible argument, restriction mapping of
MuMTV proviral DNA from BALB/cfC3H HAN's and their pri
mary outgrowths indicates that they are also a nonrandom
population.5 Therefore, the selective pressure of serial trans
plantation cannot be the sole explanation of the homogeneity
observed in the Z series outgrowths.
The differences in the integration patterns of the proviral
DNA among the five Z lines would be significant only if they
were reproducible. The restriction maps of each of the out
growth lines were identical over the 2 to 7 generations studied.
Furthermore, all transplants from a given line examined in any
one generation had identical EcoRI or BamHI patterns. These
results indicated that the integration patterns of the C3H
MuMTV DNA were stable. The fact that new patterns did not
appear with time was consistent with a concept of stable
nonrandom populations.
While the bulk of the data was consistent with nonrandom
homogeneous populations of hyperplastic cells, the Z5 lines of
hyperplastic mammary outgrowths suggested that nodules and
the resulting outgrowths were not necessarily composed of a
single stable population of cells. The Z5c, Z5c,, and Z5d
outgrowths were all developed from a single nodule. Z5c, and
5 R. D. Cardiff, D. W. Morris. T. G. Fanning, and L. J. T. Young, unpublished
observations.
3028
Z5c were developed by transplantation of tissue from the same
outgrowth. All 3 outgrowths were biologically different (1). All
3 outgrowths had unique EcoRI and BamHI C3H MuMTV
proviral DNA restriction fragments. On the other hand, all 3
lines had at least one C3H MuMTV proviral DNA fragment
which had the same size as found in another Z5 line. The
presence of common size fragments could be evidence of a
common origin with a divergent evolution of each subline.
The stability of the EcoRI and SamHI restriction maps over
many generations and the selection of 3 unique populations
from a single nodule are difficult to reconcile under a single
hypothesis. Since we are limited to an analysis of restriction
fragment size and do not have the original HAN for comparison,
we cannot distinguish between a clonal origin with divergent
evolution of the 3 sublines and the fortuitous selection of 3 of
many subpopulations. Experiments are in progress to test
these and other possible explanations.
The experiments reported here do establish that the Z series
hyperplastic mouse mammary outgrowth lines each have a
unique EcoRI and BamHI MuMTV proviral DNA restriction
pattern. Thus, the amplified C3H MuMTV proviral DNA and the
nonrandom cell populations documented previously in BALB/
cfC3H and other mammary tumors are not exclusive charac
teristics of cancer. Rather, nonrandom populations with ampli
fied MuMTV DNA, as observed with restriction mapping, can
occur at a much earlier time in neoplastic progression than was
recognized previously. This clearly implies that mammary "hyperplasias' ' are neoplastic rather than ' 'preneoplastic' ' as clas
sified by most authors (2). We propose that mammary hyperplasias are, in fact, clonal dominant premalignant
(1).
neoplasms
ACKNOWLEDGMENTS
We thank D. Mitchell, J. P. Puma, L. J. T. Young, T. S. Pratt, and J. Walls for
excellent technical assistance.
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3029
Restriction Endonuclease Studies of Hyperplastic Outgrowth
Lines from BALB/cfC3H Mouse Hyperplastic Mammary Nodules
Robert D. Cardiff, Thomas G. Fanning, David W. Morris, et al.
Cancer Res 1981;41:3024-3029.
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