Disruption of the cyclin-dependent kinase-inhibitory domain of p27 enhances growth of mice. Growth is attributed to an increase in cell number, due to increased cell proliferation, most obviously in tissues that ordinarily express p27 at the highest levels. Disruption of p27 function leads to nodular hyperplasia in the intermediate lobe of the pituitary. However, increased growth occurs without an increase in the amounts of either growth hormone or IGF-I. In addition, female mice were infertile. Luteal cell differentiation is impaired, and a disordered estrus cycle is detected. These results reflect a disturbance of the hypothalamic-pituitary-ovarian axis. The phenotypes of these mice suggest that loss of p27 causes an alteration in cell proliferation that can lead to specific endocrine dysfunction.

The mechanism by which cyclin-dependent kinase 4 (CDK4) regulates cell cycle progression is not entirely clear. Cyclin D/CDK4 appears to initiate phosphorylation of retinoblastoma protein (Rb) leading to inactivation of the S-phase-inhibitory action of Rb. However, cyclin D/CDK4 has been postulated to act in a noncatalytic manner to regulate the cyclin E/CDK2-inhibitory activity of p27Kip1 by sequestration. In this study we investigated the roles of CDK4 in cell cycle regulation by targeted disruption of the mouse CDK4 gene. CDK4؊/؊ mice survived embryogenesis and showed growth retardation and reproductive dysfunction associated with hypoplastic seminiferous tubules in the testis and perturbed corpus luteum formation in the ovary. These phenotypes appear to be opposite to those of p27-deficient mice such as gigantism and gonadal hyperplasia. A majority of CDK4 ؊/؊ mice developed diabetes mellitus by 6 weeks, associated with degeneration of pancreatic islets. Fibroblasts from CDK4 ؊/؊ mouse embryos proliferated similarly to wild-type embryonic fibroblasts under conditions that promote continuous growth. However, quiescent CDK4 ؊/؊ fibroblasts exhibited a substantial (ϳ6-h) delay in S-phase entry after serum stimulation. This cell cycle perturbation by CDK4 disruption was associated with increased binding of p27 to cyclin E/CDK2 and diminished activation of CDK2 accompanied by impaired Rb phosphorylation. Importantly, fibroblasts from CDK4 ؊/؊ p27 ؊/؊ embryos displayed partially restored kinetics of the G 0 -S transition, indicating the significance of the sequestration of p27 by CDK4. These results suggest that at least part of CDK4's participation in the rate-limiting mechanism for the G 0 -S transition consists of controlling p27 activity.In mammalian cells, the balance of growth-stimulatory and -inhibitory signals regulates the transition between proliferation and quiescence (42). Cyclin-dependent kinases (CDKs) activated by the regulatory subunits, cyclins, control cell cycle progression in all eukaryotes (21, 48, 52). Among several cyclin-CDK complexes, cyclin D-and cyclin E-dependent kinases play critical roles in regulating G 1 progression and entry into S phase. D-type cyclins bind to and activate CDK4 during early to mid-G 1 . This is followed by activation of CDK2 in complex with cyclin E during late G 1 . These two types of CDKs seem to collaborate to determine the rate of the G 1 to S transition (1,40,45). After cells enter S phase, cyclin A binds to and activates CDK2, which is required for maintenance of DNA replication (41). Three D-type cyclins (D1, D2, and D3) are expressed in tissue-specific but overlapping manners (32), whereas CDK4 and CDK2 and cyclins E and A are ubiquitously expressed. D-type cyclins also activate CDK6, a kinase closely related to CDK4 (2, 35). Although CDK6 and CDK4 are coexpressed in many cell types, it is unclear whether these two CDKs have completely overlapping functions.Cyclin D-CDK4 plays an important role in inactivating the S-phase-inhibitory action of the retinobla...

The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNAbinding domain and whose members play essential roles in cellular proliferation, differentiation, transformation, longevity, and metabolic homeostasis. Liver regeneration studies with transgenic mice demonstrated that FoxM1B regulates the onset of hepatocyte DNA replication and mitosis by stimulating expression of cell cycle genes. Here, we demonstrate that albumin-promoter-driven Cre recombinase-mediated hepatocyte-specific deletion of the Foxm1b Floxed (fl) targeted allele resulted in significant reduction in hepatocyte DNA replication and inhibition of mitosis after partial hepatectomy. Reduced DNA replication in regenerating Foxm1b ؊/؊ hepatocytes was associated with sustained increase in nuclear staining of the cyclin-dependent kinase (Cdk) inhibitor p21 Cip1 (p21) protein between 24 and 40 h after partial hepatectomy. Furthermore, increased nuclear p21 levels and reduced expression of Cdc25A phosphatase coincided with decreases in Cdk2 activation and hepatocyte progression into S-phase. Moreover, the significant reduction in hepatocyte mitosis was associated with diminished mRNA levels and nuclear expression of Cdc25B phosphatase and delayed accumulation of cyclin B1 protein, which is required for Cdk1 activation and entry into mitosis. Cotransfection studies demonstrate that FoxM1B protein directly activated transcription of the Cdc25B promoter region. Our present study shows that the mammalian Foxm1b transcription factor regulates expression of cell cycle proteins essential for hepatocyte entry into DNA replication and mitosis.knock-out mouse ͉ Cdc25A ͉ Cdc25B ͉ cyclin-dependent kinase inhibitor p21 Cip1

The cyclin-dependent kinase inhibitor p27(Kip1) is known as a negative regulator of cell-cycle progression and as a tumour suppressor. Cdk2 is the main target of p27 (refs 2, 3) and therefore we hypothesized that loss of Cdk2 activity should modify the p27(-/-) mouse phenotype. Here, we show that although p27(-/-) Cdk2(-/-) mice developed ovary tumours and tumours in the anterior lobe of the pituitary, we failed to detect any functional complementation in p27(-/-) Cdk2(-/-) double-knockout mice, indicating a parallel pathway regulated by p27. We observed elevated levels of S phase and mitosis in tissues of p27(-/-) Cdk2(-/-) mice concomitantly with elevated Cdc2 activity in p27(-/-) Cdk2(-/-) extracts. p27 binds to Cdc2, cyclin B1, cyclin A2, or suc1 complexes in wild-type and Cdk2(-/-) extracts. In addition, cyclin E binds to and activates Cdc2. Our in vivo results provide strong evidence that Cdc2 may compensate the loss of Cdk2 function.

Cyclin D1 is overexpressed in the majority of human breast cancers. We previously found that mice lacking cyclin D1 are resistant to mammary carcinomas triggered by the ErbB-2 oncogene. In this study, we investigated which function of cyclin D1 is required for ErbB-2-driven mammary oncogenesis. We report that the ability of cyclin D1 to activate cyclin-dependent kinase CDK4 underlies the critical role for cyclin D1 in breast cancer formation. We also found that the continued presence of CDK4-associated kinase activity is required to maintain breast tumorigenesis. We analyzed primary human breast cancers and found high cyclin D1 levels in a subset (approximately 25%) of ErbB-2-overexpressing tumors. We propose that this subset of breast cancer patients might benefit from inhibiting CDK4 kinase.

p21Cip1/WAF1 was the first cyclin-dependent kinase (CDK) inhibitor to be identified, as a mediator of p53 in DNA damage-induced growth arrest, cell senescence, and direct CDK regulation. p21 may also play an important role in differentiation-associated growth arrest, as its expression is augmented in many terminally differentiating cells. A general involvement of p21 in growth/differentiation control and tumor suppression has been questioned, as mice lacking p21 undergo a normal development, harbor no gross alterations in any of their organs, and exhibit no increase in spontaneous tumor development. However, a significant imbalance between growth and differentiation could be unmasked under conditions where normal homeostatic mechanisms are impaired. We report here that primary keratinocytes derived from p21 knockout mice, transformed with a ras oncogene, and injected subcutaneously into nude mice exhibit a very aggressive tumorigenic behavior, which is not observed with wild-type control keratinocytes nor with keratinocytes with a disruption of the closely related p27 gene. p21 knockout keratinocytes tested under well-defined in vitro conditions show a significantly increased proliferative potential, which is also observed but to a lesser extent with p27 knockout cells. More profound differences were found in the differentiation behavior of p21 versus p27 knockout keratinocytes, with p21 (but not p27) deficiency causing a drastic down-modulation of differentiation markers linked with the late stages of the keratinocyte terminal differentiation program. Thus, our results reveal a so far undetected role of p21 in tumor suppression, demonstrate that this function is specific as it cannot be attributed to the closely related p27 molecule, and point to an essential involvement of p21 in terminal differentiation control, which may account for its role in tumor suppression.

Cell growth and proliferation require coordinated ribosomal biogenesis and translation. Eukaryotic Initiation Factors (eIF) control translation at the rate-limiting step of initiation 1,2 . So far, only two eIFs connect extracellular stimuli to global translation rates 3 ; eIF4E acts in the eIF4F complex and regulates binding of capped mRNA to 40S subunits, downstream of growth factors 4 ; eIF2 controls loading of the ternary complex on the 40S subunit and is inhibited upon stress stimuli [5][6] . No eIFs have been found to link extracellular stimuli to the activity of the large 60S ribosomal subunit. eIF6 binds 60S ribosomes precluding ribosome joining in vitro [7][8][9] . However studies in yeasts showed that eIF6 is required for ribosome biogenesis rather than translation [10][11][12][13] . We show that mammalian eIF6 is required for efficient initiation of translation, in vivo. eIF6 null embryos are lethal at preimplantation. Heterozygous mice have 50% reduction of eIF6 levels in all tissues, and show reduced mass of hepatic and adipose tissues due to a lower number of cells and to impaired G1/S cell cycle progression. eIF6 +/− cells retain sufficient nucleolar eIF6 and normal ribosome biogenesis. The liver of eIF6 +/− mice displays an increase of 80S in polysomal profiles, indicating a defect in initiation of translation. Consistently, isolated hepatocytes have impaired insulin-stimulated translation. Heterozygous mouse embryonic fibroblasts (MEFs) recapitulate the organism phenotype and have normal ribosome biogenesis, reduced insulin-stimulated translation, and delayed G1/S phase progression. Furthermore, eIF6 +/− cells resist to oncogene-induced transformation. Thus, eIF6 is the first eIF associated with the large 60S subunit that regulates translation in response to extracellular signals.The eIF6 gene was deleted by homologous recombination using embryonic stem (ES) cell technology ( Supplementary Fig. 1a). The portion of the gene containing the first two exons and the first two introns was substituted by a cassette containing the neomycin resistance gene. The presence of the neomycin resistance cassette did not affect expression of wt eIF6 and of adjacent genes ( Supplementary Fig. 2). Germline transmission was achieved and intercrossingCorrespondence and requests for materials should be addressed to stefano.biffo@hsr.it. * These authors contributed equally Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. Table 1). The lethality of eIF6 −/− embryos is consistent with the early expression of the protein in the blastocysts ( Supplementary Fig. 1d).Heterozygous eIF6 +/− mice were viable and indistinguishable from wt counterparts up to 30 days after birth. At three months of age, heterozygous mice, independently from gender and genetic background, weighted less than their wt littermates (Fig. 1a). The head-anus length of eIF6 +/− and wt mice was identical, suggesting that the reduction of body mass in eIF6 +/− mice could be due to smaller size of specific...

Conditional deletion of the mouse Forkhead Box (Fox) m1b targeted allele in adult hepatocytes (Foxm1, previously called HFH-11B, Trident, Win, or MPP2) demonstrated that the Foxm1b transcription factor is essential for hepatocyte mitosis during liver regeneration. To determine the role of Foxm1b in liver development, we have generated Foxm1b -/- mice that deleted the Foxm1b exons encoding the winged helix DNA binding and transcriptional activation domains. Here, we show that all of the Foxm1b -/- embryos died in utero by 18.5 days postcoitum (dpc). Embryonic Foxm1b -/- livers displayed a 75% reduction in the number of hepatoblasts, resulting from diminished DNA replication and a failure to enter mitosis causing a polyploid phenotype. Reduced hepatoblast mitosis was associated with decreased protein levels of the Polo-like kinase 1 and Aurora B kinase, which phosphorylate regulatory proteins essential for orchestrating mitosis and cytokinesis. Diminished proliferation of Foxm1b -/- hepatoblasts contributed to abnormal liver development with significant reduction in the number of large hepatic veins compared to embryonic wild-type (WT) liver. Furthermore, embryonic Foxm1b -/- livers did not develop intrahepatic bile ducts, and these presumptive biliary hepatoblasts failed to express either biliary cytokeratins or nuclear levels of hepatocyte nuclear factor 1beta. These results suggest that Foxm1b is critical for hepatoblast precursor cells to differentiate toward biliary epithelial cell lineage. Finally, we used a hepatoblast-specific Cre recombinase transgene to mediate deletion of the Foxm1b fl/fl allele in the developing liver, and these embryos died in utero and exhibited diminished hepatoblast proliferation with similar abnormalities in liver morphogenesis, suggesting that the defect in liver development contributed to embryonic lethality.