The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K.

Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation1. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets2. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines3-5, we identify CR8-a cyclin-dependent kinase (CDK) inhibitor6-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.

Untimely expression of gametogenic genes in vegetative cells causes uniparental disomy.

Uniparental disomy (UPD), in which an individual contains a pair of homologous chromosomes originating from only one parent, is a frequent phenomenon that is linked to congenital disorders and various cancers. UPD is thought to result mostly from pre- or post-zygotic chromosome missegregation. However, the factors that drive UPD remain unknown. Here we use the fission yeast Schizosaccharomyces pombe as a model to investigate UPD, and show that defects in the RNA interference (RNAi) machinery or in the YTH domain-containing RNA elimination factor Mmi1 cause high levels of UPD in vegetative diploid cells. This phenomenon is not due to defects in heterochromatin assembly at centromeres. Notably, in cells lacking RNAi components or Mmi1, UPD is associated with the untimely expression of gametogenic genes. Deletion of the upregulated gene encoding the meiotic cohesin Rec8 or the cyclin Crs1 suppresses UPD in both RNAi and mmi1 mutants. Moreover, overexpression of Rec8 is sufficient to trigger UPD in wild-type cells. Rec8 expressed in vegetative cells localizes to chromosomal arms and to the centromere core, where it is required for localization of the cohesin subunit Psc3. The centromeric localization of Rec8 and Psc3 promotes UPD by uniquely affecting chromosome segregation, causing a reductional segregation of one homologue. Together, these findings establish the untimely vegetative expression of gametogenic genes as a causative factor of UPD, and provide a solid foundation for understanding this phenomenon, which is linked to diverse human diseases.

Kinase-independent function of E-type cyclins in liver cancer.

E-type cyclins (cyclins E1 and E2) are components of the core cell cycle machinery and are overexpressed in many human tumor types. E cyclins are thought to drive tumor cell proliferation by activating the cyclin-dependent kinase 2 (CDK2). The cyclin E1 gene represents the site of recurrent integration of the hepatitis B virus in the pathogenesis of hepatocellular carcinoma, and this event is associated with strong up-regulation of cyclin E1 expression. Regardless of the underlying mechanism of tumorigenesis, the majority of liver cancers overexpress E-type cyclins. Here we used conditional cyclin E knockout mice and a liver cancer model to test the requirement for the function of E cyclins in liver tumorigenesis. We show that a ubiquitous, global shutdown of E cyclins did not visibly affect postnatal development or physiology of adult mice. However, an acute ablation of E cyclins halted liver cancer progression. We demonstrated that also human liver cancer cells critically depend on E cyclins for proliferation. In contrast, we found that the function of the cyclin E catalytic partner, CDK2, is dispensable in liver cancer cells. We observed that E cyclins drive proliferation of tumor cells in a CDK2- and kinase-independent mechanism. Our study suggests that compounds which degrade or inhibit cyclin E might represent a highly selective therapeutic strategy for patients with liver cancer, as these compounds would selectively cripple proliferation of tumor cells, while sparing normal tissues.

Cyclin K dependent regulation of Aurora B affects apoptosis and proliferation by induction of mitotic catastrophe in prostate cancer.

Cyclin K plays a critical role in transcriptional regulation as well as cell development. However, the role of Cyclin K in prostate cancer is unknown. Here, we describe the impact of Cyclin K on prostate cancer cells and examine the clinical relevance of Cyclin K as a biomarker for patients with prostate cancer. We show that Cyclin K depletion in prostate cancer cells induces apoptosis and inhibits proliferation accompanied by an accumulation of cells in the G2/M phase. Moreover, knockdown of Cyclin K causes mitotic catastrophe displayed by multinucleation and spindle multipolarity. Furthermore, we demonstrate a Cyclin K dependent regulation of the mitotic kinase Aurora B and provide evidence for an Aurora B dependent induction of mitotic catastrophe. In addition, we show that Cyclin K expression is associated with poor biochemical recurrence-free survival in patients with prostate cancer treated with an adjuvant therapy. In conclusion, targeting Cyclin K represents a novel, promising anti-cancer strategy to induce cell cycle arrest and apoptotic cell death through induction of mitotic catastrophe in prostate cancer cells. Moreover, our results indicate that Cyclin K is a putative predictive biomarker for clinical outcome and therapy response for patients with prostate cancer.

SCF(Cyclin F) controls centrosome homeostasis and mitotic fidelity through CP110 degradation.

Generally, F-box proteins are the substrate recognition subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes, which mediate the timely proteolysis of important eukaryotic regulatory proteins. Mammalian genomes encode roughly 70 F-box proteins, but only a handful have established functions. The F-box protein family obtained its name from Cyclin F (also called Fbxo1), in which the F-box motif (the approximately 40-amino-acid domain required for binding to Skp1) was first described. Cyclin F, which is encoded by an essential gene, also contains a cyclin box domain, but in contrast to most cyclins, it does not bind or activate any cyclin-dependent kinases (CDKs). However, like other cyclins, Cyclin F oscillates during the cell cycle, with protein levels peaking in G2. Despite its essential nature and status as the founding member of the F-box protein family, Cyclin F remains an orphan protein, whose functions are unknown. Starting from an unbiased screen, we identified CP110, a protein that is essential for centrosome duplication, as an interactor and substrate of Cyclin F. Using a mode of substrate binding distinct from other F-box protein-substrate pairs, CP110 and Cyclin F physically associate on the centrioles during the G2 phase of the cell cycle, and CP110 is ubiquitylated by the SCF(Cyclin F) ubiquitin ligase complex, leading to its degradation. siRNA-mediated depletion of Cyclin F in G2 induces centrosomal and mitotic abnormalities, such as multipolar spindles and asymmetric, bipolar spindles with lagging chromosomes. These phenotypes were reverted by co-silencing CP110 and were recapitulated by expressing a stable mutant of CP110 that cannot bind Cyclin F. Finally, expression of a stable CP110 mutant in cultured cells also promotes the formation of micronuclei, a hallmark of chromosome instability. We propose that SCF(Cyclin F)-mediated degradation of CP110 is required for the fidelity of mitosis and genome integrity.

Concurrent deletion of cyclin E1 and cyclin-dependent kinase 2 in hepatocytes inhibits DNA replication and liver regeneration in mice.

UNLABELLED: The liver has a strong regenerative capacity. After injury, quiescent hepatocytes can reenter the mitotic cell cycle to restore tissue homeostasis. This G(0) /G(1) -S cell-cycle transition of primed hepatocytes is regulated by complexes of cyclin-dependent kinase 2 (Cdk2) with E-type cyclins (CcnE1 or CcnE2). However, single genetic ablation of either E-cyclin or Cdk2 does not affect overall liver regeneration. Here, we systematically investigated the contribution of CcnE1, CcnE2, and Cdk2 for liver regeneration after partial hepatectomy (PH) by generating corresponding double- and triple-knockout (KO) mouse mutants. We demonstrate that conditional deletion of Cdk2 alone in hepatocytes resulted in accelerated induction of CcnE1, but otherwise normal initiation of S phase in vivo and in vitro. Excessive CcnE1 did not contribute to a noncanonical kinase activity, but was located at chromatin together with components of the pre-replication complex (pre-RC), such as the minichromosome maintenance (MCM) helicase. Concomitant ablation of Cdk2 and CcnE1 in hepatocytes caused a defect in pre-RC formation and further led to dramatically impaired S-phase progression by down-regulation of cyclin A2 and cell death in vitro and substantially reduced hepatocyte proliferation and liver regeneration after PH in vivo. Similarly, combined loss of CcnE1 and CcnE2, but also the Cdk2/CcnE1/CcnE2 triple KO in liver, significantly inhibited S-phase initiation and liver mass reconstitution after PH, whereas concomitant ablation of CcnE2 and Cdk2 had no effect. CONCLUSION: In the absence of Cdk2, CcnE1 performs crucial kinase-independent functions in hepatocytes, which are capable of driving MCM loading on chromatin, cyclin A2 expression, and S-phase progression. Thus, combined inactivation of Cdk2 and CcnE1 is the minimal requirement for blocking S-phase machinery in vivo.

Truncated Cables1 causes agenesis of the corpus callosum in mice.

Agenesis of the corpus callosum (ACC) is a congenital abnormality of the brain structure. More than 60 genes are known to be involved in corpus callosum development. However, the molecular mechanisms underlying ACC are not fully understood. Previously, we produced a novel transgenic mouse strain, TAS, carrying genes of the tetracycline-inducible expression system that are not involved in brain development, and inherited ACC was observed in the brains of all homozygous TAS mice. Although ACC was probably induced by transgene insertion mutation, the causative gene and the molecular mechanism of its pathogenesis remain unclear. Here, we first performed interphase three-color fluorescence in situ hybridization (FISH) analysis to determine the genomic insertion site. Transgenes were inserted into chromosome 18 ∼12.0 Mb from the centromere. Gene expression analysis and genomic PCR walking showed that the genomic region containing exon 4 of Cables1 was deleted by transgene insertion and the other exons of Cables1 were intact. The mutant allele was designated as Cables1(TAS). Interestingly, Cables1(TAS) mRNA consisted of exons 1-3 of Cables1 and part of the transgene that encoded a novel truncated Cables1 protein. Homozygous TAS mice exhibited mRNA expression of Cables1(TAS) in the fetal cerebrum, but not that of wild-type Cables1. To investigate whether a dominant negative effect of Cables1(TAS) or complete loss of function of Cables1 gives rise to ACC, we produced Cables1-null mutant mice. ACC was not observed in Cables1-null mutant mice, suggesting that a dominant negative effect of Cables1(TAS) impairs callosal formation. Moreover, ACC frequency in Cables1(+/TAS) mice was significantly lower than that in Cables1(-/TAS) mice, indicating that wild-type Cables1 interfered with the dominant negative effect of Cables1(TAS). This study indicated that truncated Cables1 causes ACC and wild-type Cables1 contributes to callosal formation.

Deficiency of the cyclin kinase inhibitor p21(WAF-1/CIP-1) promotes apoptosis of activated/memory T cells and inhibits spontaneous systemic autoimmunity.

A characteristic feature of systemic lupus erythematosus is the accumulation of activated/memory T and B cells. These G0/G1-arrested cells express high levels of cyclin-dependent kinase inhibitors such as p21, are resistant to proliferation and apoptosis, and produce large amounts of proinflammatory cytokines. Herein, we show that ablation of p21 in lupus-prone mice allows these cells to reenter the cell cycle and undergo apoptosis, leading to autoimmune disease reduction. Absence of p21 resulted in enhanced Fas/FasL-mediated activation-induced T cell death, increased activation of procaspases 8 and 3, and loss of mitochondrial transmembrane potential. Increased apoptosis was also associated with p53 up-regulation and a modest shift in the ratio of Bax/Bcl-2 toward the proapoptotic Bax. Proliferation and apoptosis of B cells were also increased in p21-/- lupus mice. Thus, modulation of the cell cycle pathway may be a novel approach to reduce apoptosis-resistant pathogenic lymphocytes and to ameliorate systemic autoimmunity.

Regenerative response in ischemic brain restricted by p21cip1/waf1.

Neural precursor cells from adults have exceptional proliferative and differentiative capability in vitro yet respond minimally to in vivo brain injury due to constraining mechanisms that are poorly defined. We assessed whether cell cycle inhibitors that restrict stem cell populations in other tissues may participate in limiting neural stem cell reactivity in vivo. The cyclin-dependent kinase inhibitor, p21cip1/waf1 (p21), maintains hematopoietic stem cell quiescence, and we evaluated its role in the regenerative response of neural tissue after ischemic injury using the mice deficient in p21. Although steady-state conditions revealed no increase in primitive cell proliferation in p21-null mice, a significantly larger fraction of quiescent neural precursors was activated in the hippocampus and subventricular zone after brain ischemia. The hippocampal precursors migrated and differentiated into a higher number of neurons after injury. Therefore, p21 is an intrinsic suppressor to neural regeneration after brain injury and may serve as a common molecular regulator restricting proliferation among stem cell pools from distinct tissue types.

Nitric oxide signaling is disrupted in the yeast model for Batten disease.

The juvenile form of neuronal ceroid lipofuscinoses (JNCLs), or Batten disease, results from mutations in the CLN3 gene, and it is characterized by the accumulation of lipopigments in the lysosomes of several cell types and by extensive neuronal death. We report that the yeast model for JNCL (btn1-Delta) that lacks BTN1, the homologue to human CLN3, has increased resistance to menadione-generated oxidative stress. Expression of human CLN3 complemented the btn1-Delta phenotype, and equivalent Btn1p/Cln3 mutations correlated with JNCL severity. We show that the previously reported decreased levels of L-arginine in btn1-Delta limit the synthesis of nitric oxide (.NO) in both physiological and oxidative stress conditions. This defect in .NO synthesis seems to suppress the signaling required for yeast menadione-induced apoptosis, thus explaining btn1-Delta phenotype of increased resistance. We propose that in JNCL, a limited capacity to synthesize .NO directly caused by the absence of Cln3 function may contribute to the pathology of the disease.

New hippocampal neurons are not obligatory for memory formation; cyclin D2 knockout mice with no adult brain neurogenesis show learning.

The role of adult brain neurogenesis (generating new neurons) in learning and memory appears to be quite firmly established in spite of some criticism and lack of understanding of what the new neurons serve the brain for. Also, the few experiments showing that blocking adult neurogenesis causes learning deficits used irradiation and various drugs known for their side effects and the results obtained vary greatly. We used a novel approach, cyclin D2 knockout mice (D2 KO mice), specifically lacking adult brain neurogenesis to verify its importance in learning and memory. D2 KO mice and their wild-type siblings were tested in several behavioral paradigms, including those in which the role of adult neurogenesis has been postulated. D2 KO mice showed no impairment in sensorimotor tests, with only sensory impairment in an olfaction-dependent task. However, D2 KO mice showed proper procedural learning as well as learning in context (including remote memory), cue, and trace fear conditioning, Morris water maze, novel object recognition test, and in a multifunctional behavioral system-IntelliCages. D2 KO mice also demonstrated correct reversal learning. Our results suggest that adult brain neurogenesis is not obligatory in learning, including the kinds of learning where the role of adult neurogenesis has previously been strongly suggested.

Schwann cell proliferation during Wallerian degeneration is not necessary for regeneration and remyelination of the peripheral nerves: axon-dependent removal of newly generated Schwann cells by apoptosis.

Peripheral nerve injury is followed by a wave of Schwann cell proliferation in the distal nerve stumps. To resolve the role of Schwann cell proliferation during functional recovery of the injured nerves, we used a mouse model in which injury-induced Schwann cell mitotic response is ablated via targeted disruption of cyclin D1. In the absence of distal Schwann cell proliferation, axonal regeneration and myelination occur normally in the mutant mice and functional recovery of injured nerves is achieved. This is enabled by pre-existing Schwann cells in the distal stump that persist but do not divide. On the other hand, in the wild type littermates, newly generated Schwann cells of injured nerves are culled by apoptosis. As a result, distal Schwann cell numbers in wild type and cyclin D1 null mice converge to equivalence in regenerated nerves. Therefore, distal Schwann cell proliferation is not required for functional recovery of injured nerves.

Disruption of p53 in human cancer cells alters the responses to therapeutic agents.

We have examined the effects of commonly used chemotherapeutic agents on human colon cancer cell lines in which the p53 pathway has been specifically disrupted by targeted homologous recombination. We found that p53 had profound effects on drug responses, and these effects varied dramatically depending on the drug. The p53-deficient cells were sensitized to the effects of DNA-damaging agents as a result of the failure to induce expression of the cyclin-dependent kinase inhibitor p21. In contrast, p53 disruption rendered cells strikingly resistant to the effects of the antimetabolite 5-fluorouracil (5-FU), the mainstay of adjuvant therapy for colorectal cancer. The effects on 5-FU sensitivity were observed both in vitro and in vivo, were independent of p21, and appeared to be the result of perturbations in RNA, rather than DNA, metabolism. These results have significant implications for future efforts to maximize therapeutic efficacy in patients with defined genetic alterations.

Abolition of cyclin-dependent kinase inhibitor p16Ink4a and p21Cip1/Waf1 functions permits Ras-induced anchorage-independent growth in telomerase-immortalized human fibroblasts.

Human cells are more resistant to both immortalization and malignant transformation than rodent cells. Recent studies have established the basic genetic requirements for the transformation of human cells, but much of this work relied on the expression of transforming proteins derived from DNA tumor viruses. We constructed an isogenic panel of human fibroblast cell lines using a combination of gene targeting and ectopic expression of dominantly acting mutants of cellular genes. Abolition of p21(Cip1/Waf1) and p16(Ink4a) functions prevented oncogenically activated Ras from inducing growth arrest and was sufficient for limited anchorage-independent growth but not tumorigenesis. Deletion of the tumor suppressor p53 combined with abolition of p16(Ink4a) function failed to mimic the introduction of simian virus 40 large T antigen, indicating that large T antigen may target additional cellular functions. Ha-Ras and Myc cooperated only to a limited extent, but in the absence of Ras, Myc cooperated strongly with the simian virus 40 small t antigen to elicit aggressive anchorage-independent growth. The experiments reported here further define specific components of human transformation pathways.

Cyclin F disruption compromises placental development and affects normal cell cycle execution.

Human cyclin F was originally isolated as a cDNA capable of suppressing the temperature sensitivity of a Saccharomyces cerevisiae cdc4-1 mutant. Its tightly regulated expression and high conservation in the evolutionary progression from amphibians to mammals suggest that it coordinates the timing of a critical cell cycle event. The fact that it contains an F box and can form an SCF (Skp1-Cul1/Cdc53-F-box) complex in vivo further suggests that it may also function in proteolysis. To investigate the role of cyclin F in vivo, we generated mice deficient for cyclin F and conditionally deficient mice as well as mouse embryonic fibroblasts (MEFs) conditionally deficient for cyclin F. Heterozygous animals are normal and fertile, but CycF(-/-) animals, with a myriad of developmental anomalies due in large part to failures in yolk sac and chorioallantoic placentation, die around embryonic day 10.5. Tissue-specific deletion of cyclin F revealed that it was not required for the development and function of a number of different embryonic and adult tissues. In contrast, MEFs lacking cyclin F, while viable, do exhibit cell cycle defects, including reduced population-doubling time and a delay in cell cycle reentry from quiescence, indicating that cyclin F plays a role in cell cycle regulation.

Stat1-dependent, p53-independent expression of p21(waf1) modulates oxysterol-induced apoptosis.

7-Ketocholesterol (7kchol) is prominent in atherosclerotic lesions where apoptosis occurs. Using mouse fibroblasts lacking p53, p21(waf1), or Stat1, we found that optimal 7kchol-induced apoptosis requires p21(waf1) and Stat1 but not p53. Findings were analogous in a human cell system. Apoptosis was restored in Stat1-null human cells when wild-type Stat1 was restored. Phosphorylation of Stat1 on Ser(727) but not Tyr(701) was essential for optimum apoptosis. A neutralizing antibody against beta interferon (IFN-beta) blunted Ser(727) phosphorylation and apoptosis after 7kchol treatment; cells deficient in an IFN-beta receptor subunit exhibited blunted apoptosis. IFN-beta alone did not induce apoptosis; thus, 7kchol-induced release of IFN-beta was necessary but not sufficient for optimal apoptosis. In Stat1-null cells, expression of p21(waf1) was much less than in wild-type cells; introducing transient expression of p21(waf1) restored apoptosis. Stat1 and p21(waf1) were essential for downstream apoptotic events, including cytochrome c release from mitochondria and activation of caspases 9 and 3. Our data reveal key elements of the cellular pathway through which an important oxysterol induces apoptosis. Identification of the essential signaling events that may pertain in vivo could suggest targets for therapeutic intervention.

RB-dependent S-phase response to DNA damage.

The retinoblastoma tumor suppressor protein (RB) is a potent inhibitor of cell proliferation. RB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorylation during the G(1)/S transition. RB plays an essential role in the G(1) arrest induced by a variety of growth inhibitory signals. In this report, RB is shown to also be required for an intra-S-phase response to DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in Rb(+/+) but not in Rb(-/-) mouse embryo fibroblasts. Dephosphorylation of RB in S-phase cells temporally preceded the inhibition of DNA synthesis. This S-phase dephosphorylation of RB and subsequent inhibition of DNA replication was observed in p21(Cip1)-deficient cells. The induction of the RB-dependent intra-S-phase arrest persisted for days and correlated with a protection against DNA damage-induced cell death. These results demonstrate that RB plays a protective role in response to genotoxic stress by inhibiting cell cycle progression in G(1) and in S phase.

A role for both Ets and C/EBP transcription factors and mRNA stabilization in the MAPK-dependent increase in p21 (Cip-1/WAF1/mda6) protein levels in primary hepatocytes.

In primary hepatocytes and HepG2 hepatoma cells, prolonged activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway is associated with a reduction in DNA synthesis, mediated by increased expression of the cyclin-dependent kinase inhibitor protein p21 (Cip-1/WAF1/mda6) (p21). This study was performed to evaluate the contribution of transcriptional and post-transcriptional regulation in this response. Prolonged activation of the MAPK pathway in wild-type or p21 null hepatocytes caused a large decrease and increase, respectively, in DNA synthesis. Prolonged activation of the MAPK pathway in either wild-type or p21 antisense HepG2 cells also caused large decreases and increases, respectively, in DNA synthesis. MAPK signaling increased the phosphorylation of the transcription factors Ets2, C/EBPalpha, and C/EBPbeta, and rapidly increased transcription from the p21 promoter via multiple Ets- and C/EBP-elements within the enhancer region. Eight hours after MAPK activation, loss of C/EBPbeta or Ets2 function significantly reduced MAPK-stimulated transcription from the p21 promoter and abolished increased p21 protein expression. At this time, MAPK signaling increased both p21 mRNA and p21 protein stabilities that were also demonstrated to be essential for a profound increase in p21 protein levels. Thirty-six hours after MAPK activation, transcription from the p21 promoter was still significantly reduced in cells without either C/EBPbeta or Ets2 function; however, these cells were now capable of exhibiting a partial increase in p21 protein expression. In contrast, loss of C/EBPalpha function modestly reduced MAPK-stimulated transcription from the p21 promoter but strongly inhibited the ability of prolonged MAPK activation to increase protein levels of p21. This data suggested that prolonged enhancement of p21 protein levels may be under posttranscriptional control. In agreement with this hypothesis, prolonged MAPK signaling further increased p21 mRNA stability at 36 h, compared with the 8-h time point. Our data argue that MAPK signaling increased p21 promoter activity via multiple transcription factors, which alone were insufficient for a robust prolonged increase in p21 protein levels in primary hepatocytes, and that to increase p21 protein levels also required enhanced stabilization of p21 mRNA and p21 protein. Collectively, these data suggest that loss of transcription factor and mRNA/protein stabilization functions correlates with an inability of MAPK signaling to cause growth arrest versus proliferation in primary hepatocytes.

A WD repeat protein controls the cell cycle and differentiation by negatively regulating Cdc2/B-type cyclin complexes.

In the fission yeast Schizosaccharomyces pombe, p34(cdc2) plays a central role controlling the cell cycle. We recently isolated a new gene named srw1(+), capable of encoding a WD repeat protein, as a multicopy suppressor of hyperactivated p34(cdc2). Cells lacking srw1(+) are sterile and defective in cell cycle controls. When starved for nitrogen source, they fail to effectively arrest in G1 and die of accelerated mitotic catastrophe if regulation of p34(cdc2)/Cdc13 by inhibitory tyrosine phosphorylation is compromised by partial inactivation of Wee1 kinase. Fertility is restored to the disruptant by deletion of Cig2 B-type cyclin or slight inactivation of p34(cdc2). srw1(+) shares functional similarity with rum1(+), having abilities to induce endoreplication and restore fertility to rum1 disruptants. In the srw1 disruptant, Cdc13 fails to be degraded when cells are starved for nitrogen. We conclude that Srw1 controls differentiation and cell cycling at least by negatively regulating Cig2- and Cdc13-associated p34(cdc2) and that one of its roles is to down-regulate the level of the mitotic cyclin particularly in nitrogen-poor environments.

Animal models of type 2 diabetes with reduced pancreatic beta-cell mass.

Type 2 diabetes is increasingly viewed as a disease of insulin deficiency due not only to intrinsic pancreatic beta-cell dysfunction but also to reduction of beta-cell mass. It is likely that, in diabetes-prone subjects, the regulated beta-cell turnover that adapts cell mass to body's insulin requirements is impaired, presumably on a genetic basis. We still have a limited knowledge of how and when this derangement occurs and what might be the most effective therapeutic strategy to preserve beta-cell mass. The animal models of type 2 diabetes with reduced beta-cell mass described in this review can be extremely helpful (a) to have insight into the mechanisms underlying the defective growth or accelerated loss of beta-cells leading to the beta-cell mass reduction; (b) to investigate in prospective studies the mechanisms of compensatory adaptation and subsequent failure of a reduced beta-cell mass. Furthermore, these models are of invaluable importance to test the effectiveness of potential therapeutic agents that either stimulate beta-cell growth or inhibit beta-cell death.