Vertebrates express two A-type cyclins; both associate with and activate the CDK2 protein kinase. Cyclin A1 is required in the male germ line, but its molecular functions are incompletely understood. We observed specific induction of cyclin A1 expression and promoter activity after UV and ␥-irradiation which was mediated by p53. cyclin A1 ؊/؊ cells showed increased radiosensitivity. To unravel a potential role of cyclin A1 in DNA repair, we performed a yeast triple hybrid screen and identified the Ku70 DNA repair protein as a binding partner and substrate of the cyclin A1-CDK2 complex. DNA double-strand break (DSB) repair was deficient in cyclin A1؊/؊ cells. Further experiments indicated that A-type cyclins activate DNA DSB repair by mechanisms that depend on CDK2 activity and Ku proteins. Both cyclin A1 and cyclin A2 enhanced DSB repair by homologous recombination, but only cyclin A1 significantly activated nonhomologous end joining. DNA DSB repair was specific for A-type cyclins because cyclin E was ineffective. These findings establish a novel function for cyclin A1 and CDK2 in DNA DSB repair following radiation damage.The cell cycle is regulated by external signals, especially in multicellular organisms, where cell proliferation is central to growth and differentiation (34). Internal signals ensure that cell cycle transitions do not occur until all required molecular events have been completed (20). The internal signals become evident as cell cycle checkpoints upon an insult to the cell (e.g., DNA damage) which arrests the cell cycle while repair occurs (10,14). Both internal and external signals modulate the activity of the cyclin-dependent kinases (CDKs) that catalyze the ordered transitions from one phase of the cell cycle to the next. Cyclin E and cyclin A2 both associate with CDK2, and cyclin A2 is essential for DNA replication and proliferation in somatic cells (8). Embryos with a homozygous deletion of cyclin A2 are not capable of proliferative growth (28). During G 1 and S phases, the kinase activity of CDK2 associates first with cyclin E and later with cyclin A2. The timing of these processes as well as the appropriate concentration of cyclin/CDK2 complexes is crucial for successful DNA replication (8).Cyclin A1 is a second A-type cyclin that binds CDK2. Cyclin A1 is abundantly expressed in the testis and has previously been shown to be essential for entry into the metaphase of meiosis I in the male germ line in mice (38, 44). Cyclin A1 is expressed at low levels in most other tissues, but no phenotype other than male infertility has been reported for mice lacking the cyclin A1 gene (19,42). The expression of cyclin A1 in hematopoietic progenitor cells and in acute myeloid leukemia is best characterized in somatic cells (46). Surprisingly, recent microarray data suggested that cyclin A1 was transcriptionally induced following p53 activation (21).Thus, the physiological role of cyclin A1 in somatic cells remains unknown. This prompted us to analyze the mechanisms of cyclin A1 induction in somatic cel...

In higher eukaryotes, the cyclins constitute a family of proteins involved in progression through the cell cycle. The cyclin A1 gene (Ccna1) is expressed during meiosis and is required for spermatogenesis. Targeted disruption of the Ccna1 gene with a LacZ reporter gene has allowed us to study the expression pattern of this gene in more detail. We have confirmed expression in mouse pre-meiotic spermatocytes and also detected expression in the accessory olfactory bulb, hippocampus and amygdala of the adult brain. We have also found that the amount of cyclin A1 protein influences the fertility of male mice and its action is modulated by genetic background. On an outbred genetic background (129S6/SvEv 3 MF1), Ccna1 tm1Col 2 /2 animals are sterile due to spermatogenic arrest prior to the first meiotic division while Ccna1 tm1Col 1/2 mice show reduced sperm production and fertility. This is even more pronounced on an inbred genetic background (129S6/SvEv) where Ccna1 tm1Col 1 /2 male mice are sterile due to a severe reduction in the total number of sperm.

Longitudinal bone growth results from endochondral ossification, a process that requires proliferation and differentiation of chondrocytes. It has been shown that proper endochondral bone formation is critically dependent on the retinoblastoma family members p107 and p130. However, the precise functional roles played by individual E2F proteins remain poorly understood. Using both constitutive and conditional E2F1 transgenic mice, we show that ubiquitous transgene-driven expression of E2F1 during embryonic development results in a dwarf phenotype and significantly reduced postnatal viability. Overexpression of E2F1 disturbs chondrocyte maturation, resulting in delayed endochondral ossification, which is characterized by reduced hypertrophic zones and disorganized growth plates. Employing the chondrogenic cell line ATDC5, we investigated the effects of enforced E2F expression on the different phases of chondrocyte maturation that are normally required for endochondral ossification. Ectopic E2F1 expression strongly inhibits early-and late-phase differentiation of ATDC5 cells, accompanied by diminished cartilage nodule formation as well as decreased type II collagen, type X collagen, and aggrecan gene expression. In contrast, overexpression of E2F2 or E2F3a results in only a marginal delay of chondrocyte maturation, and increased E2F4 levels have no effect. These data are consistent with the notion that E2F1 is a regulator of chondrocyte differentiation.The vertebrate skeleton is formed by two distinct mechanisms of bone formation, intramembranous and endochondral ossification (23, 52). In the former, bone is generated directly from mesenchymal progenitor cells, whereas in the latter, a cartilage matrix precedes the formation of bone structures. The axial and appendicular skeletons develop through endochondral bone formation, while intramembranous ossification is mostly restricted to the skull and part of the clavicle. Endochondral ossification is especially important for longitudinal growth of the limbs, which requires the establishment of growth plates at the extreme ends of the long bones, and spatial and temporal control of chondrocyte differentiation (80). Growth plate chondrocytes form regular columns that sequentially and synchronously progress through proliferative, prehypertrophic, and terminally differentiating hypertrophic stages (13). Hypertrophic chondrocytes play a pivotal role in coordinating endochondral ossification, since they deposit the cartilage matrix that provides a template for invading osteoblasts to form trabecular bone at the site of the bone collar.The first step in the process of endochondral bone formation involves the chondrogenic differentiation of mesenchymal cells, and this is mainly regulated by transcription factors of the Sox family (Sox5, Sox6, and Sox9) (11, 67), which control the expression of cartilage-specific marker genes, including type II collagen [␣1 (II) collagen] and aggrecan genes (10,44,61). Sox Sox6 double-null mice develop severe chondrodysplasia characterized by t...

In virtually all human tumors, genetic and epigenetic alterations have been found which affect the INK4/-CYCLIN D/RB pathway, which regulates cell cycle entry and exit in normal cells. E2F transcription factors are important downstream components of this pathway, which act by controlling the expression of genes involved in DNA replication and cell cycle progression. To determine whether E2F2 deregulation promotes proliferation and tumorigenesis in vivo, we generated E2F2 transgenic mice, in which the E and murine pim1 promoter (pp) direct high expression of E2F2 in thymic epithelial cells. E-pp-E2F2 mice start to develop cytokeratin-and ER-TR4-positive cortical thymomas from the age of 20 weeks, and within 1 year, nearly all mice succumb to gross thymic epithelial tumors. General thymic morphology is largely maintained, but T cell development is perturbed in thymomas, with proportionately less CD4 ؉ CD8 ؉ double-positive thymocytes. In the first 3 months, E2F2 transgenic thymi exhibit only mild epithelial hyperplasia, and thereafter thymomas arise stochastically, probably following additional mutations. Interestingly, E-pp-E2F1 mice do not display cortical thymomas. These data argue that E2F2 promotes unscheduled cell division and oncogenic transformation of thymic epithelial cells.

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