Summary
Human cdk2 was specifically overexpressed in the mammary glands of transgenic mice using
regulatory sequences from the ovine beta-lactoglobulin gene. The cdk2 transgenic animals
did not exhibit abnormalities, and the mammary glands were normal in appearance. However,
double transgenic animals generated from crossing the nondegradable, mutant cyclin A
transgenics with animals transgenic for cdk2, a kinase partner of cyclin A, resulted in a
more pronounced phenotype than observed in either transgenic alone. In addition to the
nuclear abnormalities evident in the mutant cyclin A transgenic mammary glands, lactating
mammary glands from the bigenic animals also exhibited focal areas of hyperplasia, as well
as a greater incidence of apoptosis. These results demonstrate in vivo cooperation between
these genes in transformation and apoptotic signaling pathways.
Citation
Bortner, D.M., and Rosenberg, M.P. Overexpression of cyclin A in the mammary glands of transgenic mice results in the induction of nuclear abnormalities and increased apoptosis. Cell Growth Diff. 6:1579-1589, 1995.
Background
Orderly progression through the cell cycle occurs by the transient activation of a series
of protein kinase complexes consisting of a cyclin regulatory component and a cdk (cyclin
dependent kinase) catalytic component. Aberrant regulation of cyclin and cdk genes has
been demonstrated in various tumors and tumor cells lines, suggesting involvement of cell
cycle genes in tumorigenesis. Cyclin A plays a dual role during the cell cycle,
functioning in S phase in association with cdk2 and in mitosis in association with cdc2.
Transgenic mice overexpressing normal and stabilized versions of cyclin A in the mammary
glands have revealed potential biological functions for cyclin A in inducing
pre-neoplastic alterations and apoptosis. One of the advantages that transgenics offer is
the ability to analyze cooperativity between genes in vivo by intercrossing lines of mice.
Since cyclin A is a regulatory component of a kinase complex, efforts were undertaken to
detemine the effects of expressing the cyclin A transgene in conjunction with one of its
kinase partners, cdk2.
Transgene
Name: BLGCDK2
Target gene: BLGCDK2 contains the human cdk2 cDNA (0.8-kb BamHI fragment).
Promoter: An ovine beta-lactoglobulin promoter expression vector (pBJ41) was used
for creating transgene constructs. The vector contains 4.3-kb of 5' and 1.9-kb of 3'
flanking beta-lactoglobulin sequences.
mouse strain
B6C3F2
In the cdk2 (BLGCDK2) transgenic animals, mammary epithelial cells were normal in appearance, and lobuloalveolar and ductal development occurred normally. In bigenic animals expressing both cdk2 and a nondegradable mutant cyclin A (BLGCDK2/BLGdCYCA), mammary epithelial cells exhibited nuclear abnormalities similar to that seen in the mutant cyclin A transgenics alone. Mid-lactation bigenic mammary glands also exhibited focal areas of hyperplasia and increased numbers of apoptotic cells (4-fold over that in BLGdCYCA, and 80-fold over that in BLGCDK2 or control mammary glands). Occassionally, the bigenic females experienced lactation problems and the mammary glands appeared to have impaired lobuloalveolar development.
Mammary development
Gene expression
RNase protection analysis of lactating, bigenic mammary glands indicated expression of
both the cdk2 and truncated cyclin A transgenes. The effects of the transgenes on
endogenous mammary gene expression has not yet been investigated.
Mechanistic implications
The more pronounced phenotype evident in the bigenic animals demonstrates cooperativity
between the cdk2 and cyclin A transgenes in vivo. However, the levels of cyclin A- and
cdk2-associated kinase activity, as measured on the artificial substrate histone H1, were
not elevated in the bigenic mammary glands relative to the truncated cyclin A transgenic
mammary glands. It is possible that the more severe phenotype could be due to: 1) regional
differences in transgene expression resulting in specific areas within the mammary gland
containing elevated cyclin A/cdk2 kinase activity (consistent with the focal nature of the
hyperplasia; 2) disruption of the normal distribution of endogeneous cyclin/cdk complexes
and/or altered substrate specificities; or 3) activation of the cyclin A/cdk2 complex at
inappropriate times during the cell cycle. These issues are currently under investigation.
key words
cdk2, cyclin A, transgenic mice, apoptosis, hyperplasia, mammary gland, cell cycle
Submitted
Molecular Biology Department
Glaxo Wellcome, Inc.
Five Moore Drive
Research Triangle Park, NC 27709
Phone: (919) 483-7902
Fax: (919) 483-3777
Contact for further information
Donna M. Bortner bortner~dm@glaxo.com