Summary
By crossing mice that carry a null allele of p53 with transgenic mice that develop mammary
adenocarcinomas under the influence of a Wnt-1 transgene, we have studied the consequences
of p53 deficiency in mammary gland neoplasia. In Wnt-1 transgenic mice homozygous for the
p53 null allele, tumors appear at an earlier age than in animals heterozygous or wild-type
at the p53 locus. About half of the tumors arising in p53 tumors lacking p53 display less
fibrotic histopathology and increased genomic instability with aneuploidy, amplifications,
and deletions, as detected by karyotype analysis and comparative genomic hybridization. In
one tumor, the amplified region of chromosome 7 had an ectopically expressed int-2/FGF3
proto-oncogene, a gene known to cooperate with Wnt-1 in the production of mammary tumors.
These findings favor a model in which p53 deficiency relaxes normal restraints on
chromosomal number and organization during tumorigenesis.
Citation
Donehower, L.A., Godley, L.A., Aldaz, C.M., Pyle, R., Shi, Y.-P., Pinkel, D., Gray, J.,
Bradley, A., Medina, D., and Varmus, H.E. (1995). Deficiency of p53 accelerates mammary
tumorigenesis in Wnt-1 transgenic mice and promotes chromosomal instability. Genes &
Development 9, 882-895.
Donehower, L.A., Harvey, M., Slagle, B.L., McArthur, M.J., Montgomery, C.A., Jr., Butel, J.S., and Bradley, A. (1992). Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356:215-221.
Tsukamoto, A.S., Grosschedl, R., Guman, R.C., Parslow, T., and Varmus, H.E. (1988). Expression of the int-1 gene in transgenic mice is associated with mammary gland hyperplasia and adenocarcinomas in male and female mice. Cell 55, 619-625.
Background
The Varmus group in 1988 had shown that the Wnt-1 proto-oncogene (originally identified as
a frequently activated target gene in MMTV-induced mammary adenocarcinomas), when
ectopically expressed as a transgene in the mammary gland (driven by an MMTV LTR),
produces early mammary gland hyperplasia and mammary adenocarcinomas. We had reported the
development and characterization of a p53 knockout mouse in 1992. Mice null for p53
(p53-/-) usually develop normally but are susceptible to a wide variety of tumors at an
early age. p53-/- mice all succumbed to tumors by ten months of age, while about 50% of
p53+/- mice developed tumors by 18 months of age. Because of the diversity of tumors in
the p53-deficient mice and long time to tumor development in the heterozygotes, it was
difficult to perform mechanistic studies on the role of p53 loss in tumor progression.
Also, the p53+/+ animals developed tumors so infrequently that we didn't have adequate
controls (i.e. p53+/+ tumors which presumably developed by a p53-independent mechanism. To
facilitate mechanistic studies and obtain a model which develop a single tumor type in a
reasonable amount of time with p53+/+ controls, we crossed Harold Varmus' Wnt-1 transgenic
mice to our p53-deficient mice. The bitransgenic mice were ideal because essentially all
of the Wnt-1 transgenic p53-/-, p53+/-, and p53+/+ females developed mammary
adenocarcinomas between 2 and 9 months of age. Interestingly, all of the Wnt-1 p53-/-
females developed mammary adenocarcinomas by 15 weeks of age, while Wnt-1 p53+/- and
p53+/+ females had similar delayed appearance of tumors up to 42 weeks. For our
mechanistic studies, we chose initially to look at the role of p53 presence or absence on
genomic instability as measured by standard karyotyping and comparative genomic
hybridization. As discussed below, absence or loss of p53 profoundly increased tumor
genomic instability.
Transgene
Wnt-1, p53
mouse strain
mixed inbred: 129/Sv X SJL X C57BL/6
Both Wnt-1 transgenic females and males showed accelerated mammary tumorigenesis in the absence of p53 (Wnt-1 TG/p53-/-). Wnt-1 transgenic p53+/- and p53+/+ females showed equivalent delayed tumor kinetics compared to their p53-/- counterparts. About half of the p53+/- females showed loss of the remaining wild type p53 allele (LOH) but loss or retention of p53 in these tumors did not affect their incidence. We assessed genomic instability in tumors from the Wnt-1 p53 females and found that p53+/+ and p53+/- tumors without p53 LOH remained karyo typically stable and usually diploid compared to p53-/- and p53+/- (with LOH) tumors. The latter tumors without p53 a variety of aneuploidy patterns and DNA copy number gains and losses as measured by comparative genomic hybridization. The chromosomal gains and losses in the tumors were non-random in nature, suggesting that some of these genomic changes could be selected for during tumor progression.
Phenotype Image
Tumor Incidence in Wnt-1 p53-deficient mice.
Histology
Histology of the mammary adenocarcinoma tumor sections revealed interesting differences
between the tumors containing wild type p53 and those missing p53. Wnt-1 p53+/+ and p53+/-
(no LOH) tumors tended to be fibrotic with more organized stromal involvement. The tumor
cells also appeared more uniform and normal. In contrast, Wnt-1 p53-/- and p53+/- (LOH)
tumors appeared very anaplastic with little fibrosis and stromal involvement (see figure).
Mammary development
Mammary development was not carefully assessed in this study. However, while not reported
in the paper, Dan Medina found evidence that Wnt-1 p53-/- female mammary glands often had
inadequate branching from end buds in the young females. It seemed as if progression and
branching of the mammary gland end buds was delayed. Otherwise, Wnt-1 p53+/- and p53+/+
females had normal ductal and alveolar development except that these were hyperplastic in
appearance.
Gene expression
Gene expression was not assessed in this study. However, from previous studies, intact p53
message is not expressed in the p53 null mice. Wnt-1 expression in the mammary gland of
all mice containing the Wnt-1 transgene is at high levels both in tumor and hyperplastic
mammary glands.
Mechanistic implications
Our study shows that complete absence of p53 dramatically accelerates mammary
tumorigenesis in a model already programmed to develop mammary tumors. However, the
mechanism of tumor acceleration by p53 absence remains unclear. Interestingly, the p53+/-
tumors which lost p53 did not arise any sooner than in p53+/+ mice. This argues that loss
of p53 in this model is probably a late event which may affect tumor progression but does
not affect initiation. We also showed that loss or absence of p53 promotes genomic
instability in the Wnt-1 initiated tumors. However, since the p53+/- (LOH) tumors showed
high levels of genomic instability yet did not arise any sooner than p53+/+ tumors, it is
likely that genomic instability is a late event which affects tumor progression but not
initiation. That genomic instability plays a role in tumor progression is supported by the
non-random nature of the chromosomal gains and losses.
key words
double transgenic mice, p53, Wnt-1
Submitted by:
Larry Donehower
Division of Molecular Virology
Baylor College of Medicine
Houston, TX 77030
Phone: 713-798-3594
Fax: 713-798-3490
Contact: larryd@bcm.tmc.edu
contributed: March 1996
last update: December 1998
