Mammary Gland Hyperplasia and Carcinomas in Transgenic Mice Expressing Human Cyclin E

Summary

Transgenic mice were generated which overexpress the human cyclin E gene under the control of the ovine beta-lactoglobulin (BLG) promoter. Cyclin E overexpression in the developing secretory mammary epithelium resulted in the occurrence of papillary projections of hyperplastic cells in lactating mammary glands. Approximately ten percent of female transgenics eventually developed mammary carcinomas, with latencies ranging from 8 to 13 months. Mammary tumors contain transgene-specific cyclin E RNA and protein, and have elevated levels of cyclin E/cdk2 kinase activity.

Citation

Bortner, D. M. and Rosenberg, M. P. (1997) Induction of mammary gland hyperplasia and carcinomas in transgenic mice expressing human cyclin E. Mol. Cell. Biol. 17:453-459.

Background

Cyclin E is involved in regulating the progression of cells through G1 phase of the cell cycle, as are the D-type cyclins. The G1 class of cyclins functions together with distinct cyclin-dependent kinase partners to control rate-limiting events for the G1/S transition. Many studies have demonstrated an association between deregulated expression of cell cycle regulatory genes, in particular G1 cyclins, and cancer. In the case of cyclin E, a relationship between overexpression and alterations in cyclin E protein and human breast cancer has been established, with the severity of the alterations correlating with tumor stage and grade. In an attempt to determine whether cyclin E plays a contributing role in mammary tumorigenesis, transgenic mice expressing cyclin E in the mammary gland were generated and analyzed. Cyclin E transgenic mice exhibited hyperplastic lesions during lactation, with over 10% of the animals eventually developing mammary carcinomas, supporting a causal relationship between deregulated cyclin E expression and malignancy of mammary epithelial cells.

Mammary Phenotype

Lactating mammary glands of cyclin E transgenic mice contained areas of hyperplasia, primarily papillary projections of hyperplastic cells. Approximately 10 to 15% of postbreeding female cyclin E transgenic mice exhibited mammary gland hyperplasia, in same cases with lobuloalveolar development consistent with the histological appearance of lactation, including the hyperplastic papillary projections. Mammary adenocarcinomas have developed in three independent lines of cyclin E transgenics after a several-month latent period. Mammary tumor development occurred in over 10% of postbreeding female cyclin E transgenics, and did not appear to correspond directly to the number of pregnancies.
Histology
H&E stained mid-lactation mammary gland sections from cyclin E transgenic mice (B), in which papillary projections of hyperplastic cells are evident when compared to a non-transgenic control (A) (slide 1). Mammary gland hyperplasia is readily apparent in mammary gland sections from an 18-month old postbreeding cyclin E transgenic (D) relative to an age-matched control (C) (slide 2).
Mammary Development
Cyclin E expression was targeted to the pregnant and lactating mammary gland through the use of the BLG milk protein gene promoter. Cyclin E expression resulted in hyperplastic abnormalities in the lactating mammary gland, but did not appear to impair mammary gland development or involution.
Gene Expression
Mammary-specific expression of the cyclin E transgene was detected in lactating mammary glands of transgenic mice by RT-PCR analysis. Expression of cyclin E protein in the nuclei of transgenic mammary epithelial cells was demonstrated by immunohistochemical analysis of lactating mammary gland sections. Transgene-specific cyclin E RNA and protein were also present in mammary adenocarcinomas derived from the cyclin E transgenics, even though tumors developed in postbreeding animals at a time when the BLG promoter is not normally functional. In addition, the functional activity of cyclin E in association with cdk2 was demonstrated in mammary tumors, which contained high levels of cyclin E- and cdk2-associated kinase activity.
Mechanistic Implications
The presence of transgene-specific cyclin E RNA and protein, as well as functional cyclin E/cdk2 kinase activity in mammary tumors from three independent lines of cyclin E transgenic mice indicate that cyclin E likely plays a causal role in tumorigenic progression in this model system. This is the first direct demonstration of a causal association between the expression of cyclin E and malignancy, and supports previous studies providing correlative evidence for such an association.
Transgene
A 1.6-kb HindIII fragment containing the human cyclin E cDNA was inserted into the EcoRV site of the BLG promoter expression vector, pBJ41. pBJ41 contains 4.3-kb of 5' and 1.9-kb of 3' BLG regulatory sequences.
Mouse Strain
B6C3F2 embryos (animals backcrossed to C57BL/6J)
Keywords
cyclinE, mammary tumor, beta-lactglobulin, cell cycle, cyclin dependent kinases

contributed by

Donna M. Bortner
Molecular Biology Department
Glaxo Wellcome, Inc.
Five Moore Drive
Research Triangle Park, NC 27709
Phone: (919) 483-7902
Fax: (919) 483-3777

e-mail: bortner~dm@glaxo.com