Gadd45a-null mice generated by gene targeting exhibited several of the phenotypes characteristic of p53-deficient mice, including genomic instability, increased radiation carcinogenesis and a low frequency of exencephaly. Genomic instability was exemplified by aneuploidy, chromosome aberrations, gene amplification and centrosome amplification, and was accompanied by abnormalities in mitosis, cytokinesis and growth control. Unequal segregation of chromosomes due to multiple spindle poles during mitosis occurred in several Gadd45a -/- cell lineages and may contribute to the aneuploidy. Our results indicate that Gadd45a is one component of the p53 pathway that contributes to the maintenance of genomic stability.

MDM2 is a cellular protein that binds to and inactivates the p53 tumor suppressor protein. Although mdm2has been shown to function as an oncogene in vitro, all studies to date have assessed MDM2 activities in the presence of p53, implicating p53 inactivation in MDM2-directed transformation. To determine the role of MDM2 in the cell cycle and in tumorigenesis and whether or not this role is dependent on p53, an MDM2 minigene was expressed during gestation and lactation in the mammary gland of both wild-type p53 (p53+/+) and p53 knockout (p53-/-) mice using the bovine 3-lactoglobulin promoter. In six different transgenic mouse lines, deregulated expression of MDM2 inhibited normal development and morphogenesis of the mammary gland, and caused cellular hypertrophy and nuclear abnormalities. These abnormalities included both multinucleated cells and enlarged cells with giant nuclei. Although there were fewer epithelial cells present in the transgenic mammary gland, no apoptosis was observed. Instead, BrdU incorporation and PCNA staining showed that 12%-27% of the transgenic mammary epithelial cells were in S phase at a time when normal cells were terminally differentiated. Analysis of DNA content showed that 30%-45% of the cells were polyploid, with DNA contents up to 16N, indicating that overexpression of MDM2 caused mammary epithelial cells to undergo multiple rounds of S phase without cell division. This phenotype was similar in the p53+/+ and p53-/-background, demonstrating a role for MDM2 in the regulation of DNA synthesis that is independent of the ability of MDM2 to inhibit p53 activity. Additionally, multiple lines of BLGMDM2 transgenic mice developed mammary tumors, confirming that overproduction of MDM2 contributes to tumorigenesis in epithelial cells in vivo.

Deregulated expression of several cell cycle regulatory genes has been demonstrated to be associated with cancer. In particular, a strong correlation has been established between inappropriate cyclin E expression and human breast cancer. To determine the ability of cyclin E to play a causative role in mammary tumorigenesis, regulatory sequences from the ovine ␤-lactoglobulin gene were utilized to specifically target expression of human cyclin E to the mammary glands of pregnant and lactating mice. Lactating mammary glands of transgenic mice expressing cyclin E contained areas of hyperplasia, primarily papillary projections of hyperplastic cells, which were rarely observed in lactating glands of control mice. Over 10% of female cyclin E transgenic mice have developed mammary carcinomas, with latencies ranging from 8 to 13 months. Tumor analysis revealed the presence of transgene-specific cyclin E RNA and protein, as well as cyclin E-and cdk2-associated kinase activity, suggesting that cyclin E is likely a contributing component of tumorigenic progression in this model system.The regulation of critical transitions through the cell cycle is mediated by distinct protein kinase complexes, each composed of a cyclin regulatory and a cyclin-dependent kinase (cdk) catalytic subunit (8,35,37,41). In mammalian cells, several structural classes of cyclins (A to H) have been identified and have been demonstrated to reach maximal abundance during particular phases of the cell cycle (8, 35). Individual cyclins associate in various combinations with specific members of the cdk family, which in mammalian cells contains at least six cdks (28,35). The temporal regulation of cellular events necessary for orderly cell cycle progression is thought to be achieved by the sequential activation of different cyclin-cdk complexes throughout the cell cycle.Much evidence indicates that progression through the G 1 phase of the cell cycle is dependent upon the activity of G 1 cyclins, which include the D-type cyclins and cyclin E. The D-type cyclins reach maximal levels of expression and form functional kinase complexes with cdk4 or cdk6 during mid-G 1 phase (3, 25-27, 44, 50), whereas cyclin E is expressed and associates with cdk2 in an active complex near the G 1 /S boundary (10, 19). Inhibition of cyclin D1 by microinjection of antisense plasmids or antibodies during mid-G 1 phase prevents entry into S phase (2, 36). Similarly, microinjection of cyclin E antibodies prior to S phase inhibits the occurrence of S phase (32), as does inhibition of cdk2 activity by dominant negative or antibody microinjection methods (34,45,46). Overexpression of cyclin D1 or E accelerates progression through G 1 , reduces growth factor requirements, and diminishes cell size (31,36,39,49), while an additive effect on G 1 phase shortening is observed when both cyclins D1 and E are overexpressed in the same cell (38). These data suggest that the D-type cyclins and cyclin E are essential for progression through G 1 phase, controlling events that are rate limiting f...

The human salivary amylase genes are associated with two inserted elements, a ~/-actin-processed pseudogene and an endogenous retroviral-like element. To test the contribution of these inserted elements to tissue specificity, 25 lines of transgenic mice carrying 10 amylase constructs were established. A 1-kb fragment of AMY1C (-1003 to +2) was found to be sufficient for parotid-specific expression of a human growth hormone reporter gene. The 1-kb fragment is entirely derived from inserted sequences. Deletion from -1003 to -826 resulted in reduced levels of transgene expression and loss of tissue specificity. The fragment -1003 to -327 was sufficient to transfer parotid specificity to the thymidine kinase promoter. The data demonstrate that the functional tissue-specific promoter of human AMY1C is derived from inserted sequences and that parotid expression can be conferred by sequences derived solely from the retrovirus. A role for retrotransposition in the evolution of gene regulation is indicated by these and other recent observations.

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