Summary
Transgenic mice were generated containing wild-type or mutant human cyclin A genes
targeted to the pregnant and lactating mammary gland using regulatory sequences from the
ovine beta-lactoglobulin gene. The mutant version of cyclin A contains an amino-terminal
89 residue deletion, which encompasses the cyclin destruction box and renders the protein
nondegradable. The cyclin A transgene products were localized in the nuclei of the mammary
epithelial cells, and nuclear abnormalities were evident in the transgenic glands. The
nuclear abnormalities included karyomegaly and multinucleation and were more severe in the
mammary glands of the mutant cyclin A transgenics, which expressed a stabilized cyclin A
protein. Elevated numbers of apoptotic cells were also evident in mid-lactation transgenic
mammary glands, possibly contributing to the lack of mammary tumor development in the
cyclin A transgenic animals.
Note: an accompanying page features transgenic mice that express cdk2 and a nondegradable cyclin A mutant in their mammary glands.
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
Disruption of normal cell cycle regulation often occurs with neoplastic transformation,
and it has been suggested that cell cycle regulatory proteins may play a direct role in
the transformation process. In support of this, aberrant expression of several cell cycle
genes has been demonstrated in many types of tumors and tumor cell lines. In the case of
cyclin A, a human hepatocellular carcinoma was identified in which hepatitis B virus
integrated within cyclin A sequences and resulted in the production of an
amino-terminally-deleted, nondegradable cyclin A fusion protein. Cyclin A has also been
implicated in cellular transformation by DNA tumor viruses. While there is a strong
correlation between cell cycle gene expression and cancer, except for reports of cyclin D1
contributing to the development of B cell lymphomas and mammary tumors in transgenic mice,
the evidence that deregulated cyclin expression plays a causative role in transformation
is largely circumstantial. Transgenic mice overexpressing cyclin A proteins in the mammary
glands were generated to determine the capacity of cyclin A to function as an oncogene in
vivo and contribute to mammary tumorigenesis. In addition to wild-type cyclin A, a
nondegradable, amino-terminal mutant version of cyclin A was used to attempt to achieve
stronger cyclin A overexpression, as well as to assess the oncogenic potential of the
stabilized mutant relative to wild-type cyclin A.
Transgene
Name: BLGCYCA and BLGdCYCA
Target gene: BLGCYCA contains the human cyclin A cDNA (2.2-kb EcoRI fragment).
BLGdCYCA contains a deletion in which a 0.4-kb (EcoRI-StyI) fragment at the 5' end of the
human cyclin A cDNA was removed.
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
Mammary epithelial cells of lactating transgenic glands exhibited nuclear abnormalities (karyomegaly, multinucleation, hyperchromaticity) which were more pronounced with the nondegradable mutant cyclin A (BLGdCYCA) transgene. Increased numbers of apoptotic cells were evident during mid-lactation (9-fold in BLGCYCA, 20-fold in BLGdCYCA). Females appeared to lactate normally, and no obvious differences in lobuloalveolar or ductal development were detected.
Mammary development
Gene expression
Mammary-specific transgene expression was observed, with transgene expression detected by
RNase protection analysis in lactating mammary glands of BLGCYCA and BLGdCYCA female mice.
Immunohistochemical analysis of mid-lactation mammary gland sections revealed that
endogenous cyclin A was not detectable in control mammary glands, whereas in transgenic
glands cyclin A proteins were localized in the nuclei of mammary epithelial cells. Much
higher levels of cyclin A protein were evident in the BLGdCYCA mammary glands relative to
BLGCYCA, even though transgene expression at the RNA level was greater in the BLGCYCA
mammary glands, indicating that stabilization of cyclin A had occurred with the
amino-terminal truncation. Immunohistochemistry also revealed heterogeneity and regional
differences in transgene expression. Transgene effects on endogenous mammary gene
expression have not yet been investigated.
Mechanistic implications
The cyclin A transgenic mammary glands exhibit nuclear abnormalities that may be
suggestive of pre-neoplastic lesions, but neoplastic progression does not occur since
mammary tumors do not develop in these animals. The mammary glands of the cyclin A
transgenics also have elevated numbers of apoptotic cells, and many cells which exhibit
nuclear abnormalities stain positively for apoptosis, possibly suggesting that checkpoint
controls may be functioning properly to cause elimination of the abnormal cells. These
issues are currently being investigated.
key words
cyclin A, transgenic mice, apoptosis, mammary gland, cell cycle
Submitted
by Donna M. Bortner on January 16, 1996
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.xom