Summary
Wnt 1, formerly Int 1, was the first protooncogene implicated in mammary
tumorigenesis by MMTV insertional activation. Wnt 1 is not normally expressed in
adult mammary glands, but is expressed in defined portions of the embryonic brain and is
essential for the normal development of the central nervous system. Wnt 1 is also
expressed in early spermatids of adult males. It has been postulated that inappropriate Wnt1
expression in mammary glands may interfere with normal regulatory functions of other Wnt
gene family members that are normally expressed in mammary tissue. No role for Wnt1
homologues in human cancer has been established.
Citation
Tsukamoto AS, Grosschedl R, Guzman RC, Parslow T, Varmus HE. 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.
Shackleford GM, MacArthur CA, Kwan HC, Varmus HE. 1993. Mouse mammary tumor virus infection accelerates mammary carcinogenesis in Wnt-1 transgenic mice by insertonal activation of int-2/Fgf-3 and hst/FGF-4. Proc. Natl. Acad. Sci. USA 90:740-744.
Donehower LA, Godley LA, Aldaz CM, Pyle R, Shi YP, Pinkel D, Gray J, Bradley A, Medina D, Varmus HE. 1995. "Deficiency of p53 accelerates mammary tumorigenesis in Wnt-1 transgenic mice and promotes chromosomal instability." Genes & Development 9: 882-95.
Background
Transgene
The transgene consists of the complete coding region of long form GalTase cDNA and
contains intron 4 derived from genomic GalTase. This construct was inserted into the
EV-142 vector, containing the mouse metallothionein (MT-1) promoter and the human growth
hormone polyadenylation signal (hGH).
mouse strain
C57/Bl6 X DBA
Females homozygous for GalTase do not lactate. Heterozygotes do lactate normally (with one or two exceptions) probably reflecting different levels of transgene expression. All studies were therefore done comparing homozygous females and age-matched wild-type females. In 5-week-old transgenic virgins, the ductal tree showed reduced development and TEB1s were smaller than wild-type. Histologically, ductal structure and TEB epithelia appeared relatively normal. By 10 weeks, virgin mammary ducts appeared irregular and dilated, and secondary branching was reduced. Duct termini were enlarged; histologically these enlarged termini were distinct from TEBs. The duct epithelia of transgenic 10-week-old virgins was detached from the surrounding basement membrane. During pregnancy, transgenic mammary glands had dramatically reduced alveolar development, and alveolar epithelial cells did not appear to be secretory, compared to wild-type pregnant glands. On the day of parturition, alveolar development in transgenic glands remained low, and little to no milk was produced. Milk protein gene expression was concommitantly reduced in transgenic glands. Cultures of mammary epithelial cells derived from pregnant transgenic animals displayed abnormal interactions with a laminin-containing extracellular matrix.
Mammary development
Mammary gland development was abnormal in the transgenic mice from the onset of puberty (earlier stages have not been examined). The reduced ductal development and branching, as well as the abnormal morphology of the ducts and TEB's, became quite pronounced by 10 weeks of age in the virgin female. The defects were not due to a delay of development, but were accompanied by morphogenetic defects in the ductal epithelia, particularly in the TEB's. Proliferation rates were similar in wild-type and transgenic glands, suggesting that another mechanism, such as apoptosis, is responsible for the reduced ductal development. Morphogenetic abnormalities persist into pregnancy and at parturition, but it is not clear if these defects are secondary, and due to the reduced ductal density.
Gene expression
Since the long GalTase transgene is under the control of the metallothionein promoter,
expression is seen in multiple tissues. In fact, homozygous animals display a high rate of
neonatal lethality (up to 95%). Those animals that do survive appear to have a normal life
span, and can reproduce normally, with the exception of the lactation defect. In the
mammary gland, transgene expression, assessed by RNase protection, was high at all stages
examined (5-week-old virgin, through parturition). Western blotting detected higher levels
of GalTase protein, and GalTase enzyme assays on intact mammary epithelial cells
demonstrated a 2.3-fold increase in surface GalTase activity in transgenics compared to
wild-type.
Mechanistic implications
Cell surface GalTase has been shown to be a receptor for laminin in a variety of cell
types, and is specifically involved in cellular spreading and migration on (but not
attachment to) laminin. The cumulative in vivo and in vitro observations in this study are
consistent with the hypothesis that surface GalTase participates during normal mammary
epithelial cell interactions with the laminin-rich basal lamina, and that overexpression
of surface GalTase interferes with ductal morphogenesis. Although mammary gland
morphogenesis is reduced, we saw no evidence for a decrease in cell proliferation.
Interestingly, recent work in the Bissell and Werb labs has demonstrated that the loss of
cell-basement membrane interactions results in an increase in mammary epithelial cell
apoptosis during the normal process of involution. One possiblity to account for the
GalTase transgenic phenotype is that GalTase overexpression disturbs normal cell-basement
membrane interactions, resulting in an increase in apoptosis at an inappropriate stage of
development. Experiments to address this question and others is ongoing.
key words
galactosyltransferase, growth factors, mammary development, basement membrane, cell-matrix
interactions, transgene, metallothionein
Submitted by : Helen Hathaway on November 19, 1996
Emory University School of Medicine
Dept. of Anatomy & Cell Biology
1648 Pierce Dr.
Atlanta, GA 30322
USA
Phone: (404) 727-8158
Fax: (404) 727-6256
Contact: hhath@anatomy.emory.edu