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Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation 1 . Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets 2 . 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 lines 3-5 , we identify CR8-a cyclin-dependent kinase (CDK) inhibitor 6 -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.

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., Competing Interests: Conflict of interest statement: P.S. is a consultant and a recipient of a research grant from Novartis.

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., (© 2017 UICC.)

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.

A new member of the cyclin family cyclin Y (CCNY) is involved in the regulation of various physiological processes. In this study, the role of CCNY in energy metabolism was characterized. We found that compared with wild-type (WT) mice, Ccny knockout (KO) mice had both lower body weight and lower fat content. The Ccny KO mice also had a higher metabolic rate, resisted the stress of a high-fat diet, and were sensitive to calorie restriction. The expression levels of UCP1 and PGC1α were significantly higher in the brown adipose tissue (BAT) of the Ccny KO mice than that of the WT littermate controls, whereas there was no significant difference in BAT weight between the WT and the Ccny KO mice. In addition, the down-regulation of Ccny resulted in suppression of white adipocyte differentiation both in vivo and in vitro, while the expression of Ccny was up-regulated by C/EBPα. Furthermore, both hepatocytes and HepG2 cells that were depleted of Ccny were insensitive to insulin stimulation, consistent with the significant inhibition of insulin sensitivity in the liver of the Ccny KO mice, but no significant changes in WAT and muscle, indicating that CCNY is involved in regulating the hepatic insulin signaling pathway. The hepatic insulin resistance generated by Ccny depletion resulted in down-regulation of the sterol-regulatory element-binding protein (SREBP1) and fatty acid synthase (FASN). Together, these results provide a new link between CCNY and lipid metabolism in mice, and suggest that inhibition of CCNY may offer a therapeutic approach to obesity and diabetes.

Cyclin J (CycJ) is a poorly characterized member of the Cyclin superfamily of cyclin-dependent kinase regulators, many of which regulate the cell cycle or transcription. Although CycJ is conserved in metazoans its cellular function has not been identified and no mutant defects have been described. In Drosophila, CycJ transcript is present primarily in ovaries and very early embryos, suggesting a role in one or both of these tissues. The CycJ gene (CycJ) lies immediately downstream of armitage (armi), a gene involved in the Piwi-associated RNA (piRNA) pathways that are required for silencing transposons in the germline and adjacent somatic cells. Mutations in armi result in oogenesis defects but a role for CycJ in oogenesis has not been defined. Here we assessed oogenesis in CycJ mutants in the presence or absence of mutations in armi or other piRNA pathway genes. CycJ null ovaries appeared normal, indicating that CycJ is not essential for oogenesis under normal conditions. In contrast, armi null ovaries produced only two egg chambers per ovariole and the eggs had severe axis specification defects, as observed previously for armi and other piRNA pathway mutants. Surprisingly, the CycJ armi double mutant failed to produce any mature eggs. The double null ovaries generally had only one egg chamber per ovariole and the egg chambers frequently contained an overabundance of differentiated germline cells. Production of these compound egg chambers could be suppressed with CycJ transgenes but not with mutations in the checkpoint gene mnk, which suppress oogenesis defects in armi mutants. The CycJ null showed similar genetic interactions with the germline and somatic piRNA pathway gene piwi, and to a lesser extent with aubergine (aub), a member of the germline-specific piRNA pathway. The strong genetic interactions between CycJ and piRNA pathway genes reveal a role for CycJ in early oogenesis. Our results suggest that CycJ is required to regulate egg chamber production or maturation when piRNA pathways are compromised., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

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.

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., (© 2013 by the American Association for the Study of Liver Diseases.)

Mitotic catastrophe occurs when cells enter mitosis with damaged DNA or excess centrosomes. Cells overexpressing the centrosome protein CP110 or depleted of cyclin F, which targets CP110 for destruction, have more than two centrosomes and undergo mitotic catastrophe. Our studies show centrosome reduplication and mitotic catastrophe in osteosarcoma cells inducibly expressing a p27Kip1 mutant (termed p27K) that binds cyclins but not cyclin-dependent kinases (CDKs). p27K inhibited cell proliferation but not CDK activity or cell cycle progression. It did not induce apoptosis; however, cells expressing p27K had more than two centrosomes and, indicative of mitotic catastrophe, irregularly shaped nuclei or multiple micronuclei. p27K interacted with cyclin F in vivo (as did endogenous p27Kip1) and displaced cyclin F from CP110. Depletion of CP110 rescued p27K-expressing cells from centrosome reduplication and mitotic catastrophe. Collectively, our data show that p27Kip1 can perturb mitosis and suggest that it does so by sequestering cyclin F, which prevents its interaction with and the subsequent degradation of CP110, ultimately resulting in centrosome reduplication, mitotic catastrophe and abrogation of cell proliferation.

Virus-like particles (VLPs) of bacteriophage MS2 possess numerous features that make them well-suited for use in targeted delivery of therapeutic and imaging agents. MS2 VLPs can be rapidly produced in large quantities using in vivo or in vitro synthesis techniques. Their capsids can be modified in precise locations via genetic insertion or chemical conjugation, facilitating the multivalent display of targeting ligands. MS2 VLPs also self-assemble in the presence of nucleic acids to specifically encapsidate siRNA and RNA-modified cargos. Here we report the use of MS2 VLPs to selectively deliver nanoparticles, chemotherapeutic drugs, siRNA cocktails, and protein toxins to human hepatocellular carcinoma (HCC). MS2 VLPs modified with a peptide (SP94) that binds HCC exhibit a 10(4)-fold higher avidity for HCC than for hepatocytes, endothelial cells, monocytes, or lymphocytes and can deliver high concentrations of encapsidated cargo to the cytosol of HCC cells. SP94-targeted VLPs loaded with doxorubicin, cisplatin, and 5-fluorouracil selectively kill the HCC cell line, Hep3B, at drug concentrations <1 nM, while SP94-targeted VLPs that encapsidate a siRNA cocktail, which silences expression of cyclin family members, induce growth arrest and apoptosis of Hep3B at siRNA concentrations <150 pM. Impressively, MS2 VLPs, when loaded with ricin toxin A-chain (RTA) and modified to codisplay the SP94 targeting peptide and a histidine-rich fusogenic peptide (H5WYG) that promotes endosomal escape, kill virtually the entire population of Hep3B cells at an RTA concentration of 100 fM without affecting the viability of control cells. Our results demonstrate that MS2 VLPs, because of their tolerance of multivalent peptide display and their ability to specifically encapsidate a variety of chemically disparate cargos, induce selective cytotoxicity of cancer in vitro and represent a significant improvement in the characteristics of VLP-based delivery systems.

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.

The Drosophila gene CG14939 encodes a member of a highly conserved family of cyclins, the Y-type cyclins, which have not been functionally characterized in any organism. Here we report the generation and phenotypic characterization of a null mutant of CG14939, which we rename Cyclin Y (CycY). We show that the null mutant, CycY(E8), is homozygous lethal with most mutant animals arresting during pupal development. The mutant exhibits delayed larval growth and major developmental defects during metamorphosis, including impaired gas bubble translocation, head eversion, leg elongation, and adult tissue growth. Heat-shock-induced expression of CycY at different times during development resulted in variable levels of rescue, the timing of which suggests a key function for zygotic CycY during the transition from third instar larvae to prepupae. CycY also plays an essential role during embryogenesis since zygotic null embryos from null mothers fail to hatch into first instar larvae. We provide evidence that the CycY protein (CycY) interacts with Eip63E, a cyclin-dependent kinase (Cdk) for which no cyclin partner had previously been identified. Like CycY, the Eip63E gene has essential functions during embryogenesis, larval development, and metamorphosis. Our data suggest that CycY/Eip63E form a cyclin/Cdk complex that is essential for several developmental processes.

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.

The positive transcription elongation factor b (P-TEFb) is essential for the elongation of transcription and cotranscriptional processing by RNA polymerase II. In mammals, it contains predominantly the C-type cyclin cyclin T1 (CycT1) or CycT2 and cyclin-dependent kinase 9 (Cdk9). To determine if these cyclins have redundant functions or affect distinct sets of genes, we genetically inactivated the CycT2 gene (Ccnt2) using the beta-galactosidase-neomycin gene (beta-geo) gene trap technology in the mouse. Visualizing beta-galactosidase during mouse embryogenesis revealed that CycT2 is expressed abundantly during embryogenesis and throughout the organism in the adult. This finding was reflected in the expression of CycT2 in all adult tissues and organs. However, despite numerous matings of heterozygous mice, we observed no CycT2(-/-) embryos, pups, or adult mice. This early lethality could have resulted from decreased expression of critical genes, which were revealed by short interfering RNAs against CycT2 in embryonic stem cells. Thus, CycT1 and CycT2 are not redundant, and these different P-TEFb complexes regulate subsets of distinct genes that are important for embryonic development.

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.

AIRE is a transcriptional activator that directs the ectopic expression of many tissue-specific genes in medullary thymic epithelial cells, which plays an important role in the negative selection of autoreactive T cells. However, its mechanism of action remains poorly understood. In this study, we found that AIRE regulates the step of elongation rather than initiation of RNA polymerase II. For these effects, AIRE bound and recruited P-TEFb to target promoters in medullary thymic epithelial cells. In these cells, AIRE activated the ectopic transcription of insulin and salivary protein 1 genes. Indeed, by chromatin immunoprecipitation, we found that RNA polymerase II was already engaged on these promoters but was unable to elongate in the absence of AIRE. Moreover, the genetic inactivation of cyclin T1 from P-TEFb abolished the transcription of AIRE-responsive genes and led to lymphocytic infiltration of lacrimal and salivary glands in the CycT1-/- mouse. Our findings reveal critical steps by which AIRE regulates the transcription of genes that control central tolerance in the thymus.

Recent studies have shown that cell cycle inhibitors encoded by the Ink4a gene locus constrain the self-renewing activity of adult stem cells of the hematopoietic and nervous systems. Here we report that knockout (KO) of the Cables1 [cyclin-dependent kinase (CDK)-5 and ABL enzyme substrate 1] cell cycle-regulatory gene in mice has minimal to no effect on hematopoietic stem cell (HSC) dynamics. However, female Cables1-null mice exhibit a significant expansion of germ cell (oocyte) numbers throughout adulthood. This is accompanied by a dramatic elevation in the number of atretic immature oocytes within the ovaries and an increase in the incidence of degenerating oocytes retrieved following superovulation of CABLES1-deficient females. These outcomes are not observed in mice lacking p16INK4a alone or both p16INK4a and p19ARF. These data support recent reports that adult female mice can generate new oocytes and follicles but the enhancement of postnatal oogenesis by Cables1 KO appears offset by a reduction in oocyte quality, as reflected by increased elimination of these additional germ cells via apoptosis. This work also reveals cell lineage specificity with respect to the role that specific CDK-interacting proteins play in restraining the activity of adult germline versus somatic stem cells.

Current understanding of the integration of cell division and expansion in the development of plant lateral organs such as leaves is limited. Cell number is established during a mitotic phase, and subsequent growth into a mature organ relies primarily on cell expansion accompanied by endocycles. Here we show that the three Arabidopsis cyclin D3 (CYCD3) genes are expressed in overlapping but distinct patterns in developing lateral organs and the shoot meristem. Triple loss-of-function mutants show that CYCD3 function is essential neither for the mitotic cell cycle nor for morphogenesis. Rather, analysis of mutant and reciprocal overexpression phenotypes shows that CYCD3 function contributes to the control of cell number in developing leaves by regulating the duration of the mitotic phase and timing of the transition to endocycles. Petals, which normally do not endoreduplicate, respond to loss of CYCD3 function with larger cells that initiate endocycles. The phytohormone cytokinin regulates cell division in the shoot meristem and developing leaves and induces CYCD3 expression. Loss of CYCD3 impairs shoot meristem function and leads to reduced cytokinin responses, including the inability to initiate shoots on callus, without affecting endogenous cytokinin levels. We conclude that CYCD3 activity is important for determining cell number in developing lateral organs and the relative contribution of the alternative processes of cell production and cell expansion to overall organ growth, as well as mediating cytokinin effects in apical growth and development.

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.

Peritoneal B-1a cells differ from splenic B-2 cells in the molecular mechanisms that control G(0)-S progression. In contrast to B-2 cells, cyclin D2 is up-regulated in a rapid and transient manner in phorbol ester (PMA)-stimulated B-1a cells, whereas cyclin D3 does not accumulate until late G(1) phase. This nonoverlapping expression of cyclins D2 and D3 suggests distinct functions for these proteins in B-1a cells. To investigate the contribution of cyclin D3 in the proliferation of B-1a cells, we transduced p16(INK4a) peptidyl mimetics (TAT-p16) into B-1a cells before cyclin D3 induction to specifically block cyclin D3-cyclin-dependent kinase 4/6 assembly. TAT-p16 inhibited DNA synthesis in B-1a cells stimulated by PMA, CD40L, or LPS as well as endogenous pRb phosphorylation by cyclin D-cyclin-dependent kinase 4/6. Unexpectedly, however, cyclin D3-deficient B-1a cells proliferated in a manner similar to wild-type B-1a cells following PMA or LPS stimulation. This was due, at least in part, to the compensatory sustained accumulation of cyclin D2 throughout G(0)-S progression. Taken together, experiments in which cyclin D3 was inhibited in real time demonstrate the key role this cyclin plays in normal B-1a cell mitogenesis, whereas experiments with cyclin D3-deficient B-1a cells show that cyclin D2 can compensate for cyclin D3 loss in mutant mice.

Plant cells frequently undergo endoreduplication, a process in which chromosomal DNA is successively duplicated in the absence of mitosis. It has been proposed that endoreduplication is regulated at its entry by mitotic cyclin-dependent kinase activity. However, the regulatory mechanisms for its termination remain unclear, although plants tightly control the ploidy level in each cell type. In the process of searching for regulatory factors of endoreduplication, the promoter of an Arabidopsis thaliana cyclin A gene, CYCA2;3, was revealed to be active in developing trichomes during the termination period of endoreduplication as well as in proliferating tissues. Taking advantage of the situation that plants encode highly redundant cyclin A genes, we were able to perform functional dissection of CYCA2;3 using null mutant alleles. Null mutations of CYCA2;3 semidominantly promoted endocycles and increased the ploidy levels achieved in mature organs, but they did not significantly affect the proportion of cells that underwent endoreduplication. Consistent with this result, expression of the CYCA2;3-green fluorescent protein fusion protein restrained endocycles in a dose-dependent manner. Moreover, a mutation in the destruction box of CYCA2;3 stabilized the fusion protein in the nuclei and enhanced the restraint. We conclude that CYCA2;3 negatively regulates endocycles and acts as a key regulator of ploidy levels in Arabidopsis endoreduplication.

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.

Cyclin G-associated kinase (GAK), also known as auxilin 2, is a potential regulator of clathrin-mediated membrane trafficking. It possesses a kinase domain at its N-terminus that can phosphorylate the clathrin adaptors AP-1 and AP-2 in vitro. The GAK C-terminus can act as a cochaperaone in vitro for Hsc70, a heat-shock protein required for clathrin uncoating. Here we show that the specificity of GAK is very similar to that of adaptor-associated kinase 1, another mammalian adaptor kinase. We used siRNA to investigate GAK's in vivo function. We discovered that early stages of clathrin-mediated endocytosis (CME) were partially inhibited when GAK expression was knocked down. This defect was specifically caused by GAK knockdown because it could be rescued by expressing a rat GAK gene that could not be silenced by one of the siRNAs. To identify the GAK activity required during CME, we mutated the kinase domain and the J domain of the rat gene. Only GAK with a functional J domain could rescue the defect, suggesting that GAK is important for clathrin uncoating. Furthermore, we demonstrated that GAK plays a role in the clathrin-dependent trafficking from the trans Golgi network.

Cyclin G-associated kinase (GAK/auxilin 2), the ubiquitous form of the neuronal-specific protein auxilin 1, is an essential cofactor for the Hsc70-dependent uncoating of clathrin-coated vesicles. We have now investigated the effect of knocking down GAK in HeLa cells by vector-based small hairpin RNA. Functionally, depletion of GAK caused a marked decrease in internalization of both transferrin and epidermal growth factor and altered mannose 6-phosphate receptor trafficking, but had little effect on the recycling of transferrin receptor back to the plasma membrane. Structurally, depletion of GAK caused a marked reduction in perinuclear clathrin associated with the trans-Golgi network and in the number of clathrin-coated pits on the plasma membrane, and reduced clathrin exchange on the few clathrin-coated pits that remained. Surprisingly, while clathrin depletion does not prevent adaptors from assembling on the membrane, depletion of GAK caused a dramatic reduction in AP2 and epsin on the plasma membrane and AP1 and GGA at the trans-Golgi network. A similar effect was caused by expression of a dominant negative Hsp70 mutant. These results suggest that GAK, in conjunction with Hsc70, not only uncoats clathrin-coated vesicles and induces clathrin exchange on clathrin-coated pits, but also mediates binding of clathrin and adaptors to the plasma membrane and the trans-Golgi network.

RNA interference (RNAi), a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), can be used not only as a research tool but also as a therapeutic strategy for viral infection. We demonstrated that intracellular expression of short hairpin RNA (shRNA) targeting human cyclin T1 (hCycT1), a cellular factor essential for transcription of messenger and genomic RNAs from the long terminal repeat promoter of provirus of human immunodeficiency virus type 1 (HIV-1), could effectively suppress the replication of HIV-1. We also showed that downregulation of hCycT1 did not cause apoptotic cell death, therefore, targeting cellular factor hCycT1 by shRNAs may provide an attractive approach for genetic therapy of HIV-1 infection in the future.

The myogenic factor MyoD induces the expression of the cdk inhibitor p21 to promote cell cycle withdrawal in differentiating myoblasts. Although the cdk inhibitor p57 is also highly expressed in skeletal muscle and is thought to redundantly control myogenesis, little is known about its regulation, that has been suggested to be independent of MyoD. Here we show, for the first time, that MyoD is capable to induce the expression of p57. Intriguingly, this ability is restricted to cells lacking p21, suggesting that the two cdk inhibitors may be expressed in different muscle cell lineages. We also suggest that the functions of p21 and p57 in myoblast cells are only in part redundant. In fact, while the two cdk inhibitors play a similar role in cells undergoing G1 arrest during MyoD-induced differentiation, p57 does not replace p21 in cells escaping G1 arrest and undergoing MyoD-induced apoptosis. This difference can be ascribed both to a different subcellular localization and to a differential ability of the two cdk inhibitors to interact with cell cycle regulators., (Copyright 2004 Wiley-Liss, Inc.)

We have demonstrated previously that interferon (IFN)-gamma sensitizes human colon carcinoma cell lines to the cytotoxic effects of 5-fluorouracil combined with leucovorin and to the thymidylate synthase inhibitor, ZD9331, dependent on thymineless stress-induced DNA damage, independent of p53. Here we demonstrate that the cyclin-dependent kinase (CDK) inhibitor p21(Cip1) regulates thymineless stress-induced cytotoxicity in these cells. HCT116 wild-type (wt) and p53-/- cells underwent apoptosis and loss in clonogenic survival when exposed to ZD9331, whereas p21Cip1-/- cells were resistant. In contrast, IFN-gamma induced marked cytotoxicity in p21Cip1-/- cells only. ZD9331 induced p21Cip1 up-regulation in all of the cell lines examined, as did thymidine deprivation in thymidylate synthase-deficient (thymidylate synthase-) cells. Furthermore, selective induction of p21Cip1 in RKO was sufficient to induce apoptosis. P21Cip1, cdk1, cdk2, and cyclin E mRNA expression increased coincident with S-phase accumulation in HT29 cells treated with ZD9331 or 5fluorouracil/leucovorin, as demonstrated by cDNA microarray analyses. Cell cycle analyses revealed that HCT116 wt and p21Cip1 -/- cells accumulated in S phase within 24 h of ZD9331 exposure; however, wt cells exited S-phase more rapidly, where apoptosis occurred before mitosis, either in late S or G2. Finally, the CDK inhibitor roscovitine potentiated the cytotoxic activity of ZD9331 in both wt and p21Cip1-/- cells, strongly suggesting a role for p21Cip1-dependent CDK inhibition in cytotoxicity induced by thymidylate synthase inhibition. In summary, p21Cip1 positively regulates the cytotoxic action of thymidylate synthase inhibitors, negatively regulates the cytotoxic action of IFN-gamma, and enhances S-phase exit after thymineless stress, possibly via interaction with CDK-cyclin complexes.

Computerized video time lapse (CVTL) microscopy was used to observe cellular events induced by ionizing radiation (10-12 Gy) in nonclonogenic cells of the wild-type HCT116 colorectal carcinoma cell line and its three isogenic derivative lines in which p21 (CDKN1A), 14-3-3sigma or both checkpoint genes (double-knockout) had been knocked out. Cells that fused after mitosis or failed to complete mitosis were classified together as cells that underwent mitotic catastrophe. Seventeen percent of the wild-type cells and 34-47% of the knockout cells underwent mitotic catastrophe to enter generation 1 with a 4N content of DNA, i.e., the same DNA content as irradiated cells arrested in G(2) at the end of generation 0. Radiation caused a transient division delay in generation 0 before the cells divided or underwent mitotic catastrophe. Compared with the division delay for wild-type cells that express CDKN1A and 14-3-3sigma, knocking out CDKN1A reduced the delay the most for cells irradiated in G(1) (from approximately 15 h to approximately 3- 5 h), while knocking out 14-3-3sigma reduced the delay the most for cells irradiated in late S and G(2) (from approximately 18 h to approximately 3-4 h). However, 27% of wild-type cells and 17% of 14-3-3sigma(-/-) cells were arrested at 96 h in generation 0 compared with less than 1% for CDKN1A(-/-) and double-knockout cells. Thus expression of CDKN1A is necessary for the prolonged delay or arrest in generation 0. Furthermore, CDKN1A plays a crucial role in generation 1, greatly inhibiting progression into subsequent generations of both diploid cells and polyploid cells produced by mitotic catastrophe. Thus, in CDKN1A-deficient cell lines, a series of mitotic catastrophe events occurred to produce highly polyploid progeny during generations 3 and 4. Most importantly, the polyploid progeny produced by mitotic catastrophe events did not die sooner than the progeny of dividing cells. Death was identified as loss of cell movement, i.e. metabolic activity. Thus mitotic catastrophe itself is not a direct mode of death. Instead, apoptosis during interphase of both uninucleated and polyploid cells was the primary mode of death observed in the four cell types. Knocking out either CDKN1A or 14-3-3sigma increased the amount of cell death at 96 h, from 52% to approximately 70%, with an even greater increase to 90% when both genes were knocked out. Thus, in addition to effects of CDKN1A and 14-3-3sigma expression on transient cell cycle delay, CDKN1A has both an anti-proliferative and anti-apoptosis function, while 14-3-3sigma has only an anti-apoptosis function. Finally, the large alterations in the amounts of cell death did not correlate overall with the small alterations in clonogenic survival (dose-modifying ratios of 1.05-1.13); however, knocking out CDKN1A resulted in a decrease in arrested cells and an increase in survival, while knocking out 14-3-3sigma resulted in an increase in apoptosis and a decrease in survival.

The link between mesangial cell (MC) proliferation and migration during glomerular repair in the experimental mesangial proliferative glomerulonephritis suggests that cell cycle regulation and cell migration require similar pathways, such as cell cycle proteins. The immunosuppressant RAD inhibits mesangial cell (MC) proliferation via G1/S arrest. Moreover, RAD dramatically impairs glomerular healing in the anti-Thy1 model. We tested the hypothesis that RAD alters MC migration in vitro and that this effect was mediated by the CDK-inhibitors p21(CIP1) and p27(KIP1). Using a modified Boyden chamber in vitro migration assay, our results showed that RAD dose dependently (1-50 nM) inhibited fibronectin-induced chemotaxis in wild-type (wt) MC. RAD treatment prevented the decrease in p27(KIP1) induced by mitogenic growth factors, but had no effect on p21(CIP1) by Western blot analysis. The antimigratory effect of RAD in wt MC was substantially dependent on p27(KIP1), but not p21(CIP1), since the inhibitory effects of 1-10 nM RAD on MC migration were similar in p21(CIP1) deficient and wild-type MC. The effect of RAD on MC migration was also examined in the anti-Thy1 model by BrdU-labeling of proliferating MC on day 3 that typically repopulate the glomerulus from the hilus. A control biopsy on day 3 was taken to define the starting point prior to the initiation of RAD (3 mg/kg or placebo). MC migration was determined on day 7 by measuring the distances of BrdU-labeled MC (OX-7+/BrdU+cells) from the glomerular hilus using computerized morphometry. RAD significantly reduced the migratory response of BrdU-labeled MC compared to controls. We conclude that the immunosuppressant RAD effectively inhibits MC migration in vivo and in vitro thereby limiting the normal glomerular repair process after severe injury. Moreover, RAD-induced inhibition of MC migration in vitro is partially mediated by the CDK-inhibitor p27(KIP1), but not p21(CIP1).

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.

To study the regulation of cyclin-dependent kinase (CDK) activity during mitotic exit in mammalian cells, we constructed murine cell lines that constitutively express a stabilized mutant of cyclin A (cyclin A47). Even though cyclin A47 was expressed throughout mitosis and in G1 cells, its associated CDK activity was inactivated after the transition from metaphase to anaphase. Cyclin A47 associated with both p21 and p27 during mitotic exit, implicating these proteins in CDK inactivation. However, cyclin A47 was fully inhibited during the M-to-G1 transition in p21(-/-) p27(-/-) fibroblasts. Also, the CDKs associated with cyclin A47 were not inactivated by phosphorylation at tyrosines. The protein responsible for CDK inactivation during mitotic exit in p21/p27 null cells was the Rb family member, p107. p107 bound to cyclin A47 when p21 and p27 were absent, and cyclin A47-CDK activity was not inactivated during the M-to-G1 transition in p21(-/-) p27(-/-) p107(-/-) null fibroblasts. Enforced expression of cyclin A in cells lacking all three CDK inhibitors induced rapid tetraploidization, indicative of mitotic failure/endoreduplication. We concluded that cyclin proteolysis and CDK inhibitors constitute redundant pathways that control cyclin A-CDK activity during mitotic exit in mammalian cells and that loss of these pathways can cause genetic instability.

Chinese hamster ovary cells have been engineered to inducibly over-express the p21(CIP1) cyclin-dependent kinase inhibitor, to achieve cell cycle arrest and increase cell productivity. In p21(CIP1)-arrested cells production of antibody from a stably integrated lgG4 gene, was enhanced approximately fourfold. The underlying physiological basis for enhanced productivity was investigated by measuring a range of cellular and metabolic parameters. Interestingly, the average cell volume of arrested cells was approximately fourfold greater than that of proliferating cells. This was accompanied by significant increases in mitochondrial mass, mitochondrial activity, and ribosomal protein S6 levels. Our results suggest that p21(CIP1)-induced cell cycle arrest uncouples cell growth from cell-cycle progression, and provides new insight into how improved productivity can be achieved in a cell line commonly used for large-scale production of pharmaceutical proteins., (Copyright 2004 Wiley Periodicals, Inc.)

p21(Cip1) (p21) controls cell cycle progression and apoptosis in mature endothelial cells (ECs) and regulates size and cycling of the hematopoietic progenitor cell pool. Because circulating endothelial progenitor cells (EPCs) contribute to postnatal neovascularization in addition to mature ECs, we investigated the regulation of ECs and EPCs in p21-deficient mice. Mature aortic EC proliferation was increased in homozygous p21(-/-) and heterozygous p21(+/-) mice, in which p21 protein levels are reduced to one third of wild-type (WT). In contrast, apoptosis sensitivity was increased by 3.5-fold only in p21(-/-), but not in p21(+/-) mice. Consistently, in vivo apoptosis of ECs within areas of neovascularization was elevated in p21(-/-) but not in p21(+/-) mice. EPC numbers were elevated 2-fold in p21(-/-) mice compared with WT (P<0.001), and clonal expansion capacity of EPCs was increased from 25+/-4 (WT) to 57+/-8 colony-forming units in p21(-/-) mice (P<0.005). EPC numbers and expansion were likewise increased in p21(+/-) mice. As the integrative endpoint, in vivo neovascularization reflecting all p21-affected parameters was increased over WT only in p21(+/-) (P<0.001), but not in p21(-/-) mice. In conclusion, reduced p21 protein levels of mice lacking one p21 allele are associated with increased proliferation of ECs and EPCs, whereas survival of ECs to apoptotic stimuli in vitro and in vivo is not impaired. Under these conditions, neovascularization was increased. In contrast, complete p21 deficiency did not result in an increased neovascularization despite increased mature EC and EPC proliferation. This may be due to the sensitization of ECs against apoptosis.

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.

Using Cre-Lox technology to inducibly delete Rb from wild-type, p21- and/or p53-deficient primary hepatocytes, we investigated the role of p53, p21 and pRb in the regulation of liver cell proliferation, polyploidization and death. These cellular decisions are critical to maintaining liver cell replacement in disease, and in determining the likelihood of carcinogenesis in chronic liver injury. Clearly, the present study shows a complex interplay between p53, p21 and pRb, which regulates the likelihood of hepatocytes stimulated from quiescence, to proliferate, undergo polyploidy or die. It reveals that these proteins act both in concert and independently, demonstrating that a small set of key cellular players is common to diverse cell decisions of fundamental importance to disease.

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.

The cyclin-dependent kinase inhibitor p21WAF1/Cip1 and Survivin enhance granulocyte macrophage colony-forming unit (CFU-GM) cell cycle and proliferation and have been implicated as antiapoptotic proteins. We investigated the relationships between p21 and Survivin in primary CFU-GM and c-kit+, lineage-negative (Lin-) cells and demonstrate p21-dependent and -independent pathways whereby Survivin regulates progenitor cell proliferation. Ectopic Survivin enhanced p21+/+ CFU-GM formation and expansion of c-kit+, Lin- cells, whereas p21 gene loss abrogated these effects, indicating a p21 requirement. A dominant-negative form of Survivin and p21 gene deletion accelerated the loss of CFU-GM upon growth factor deprivation, and wild-type Survivin overexpression inhibited apoptosis of p21+/+ CFU-GM and c-kit+, Lin- cells but not p21-/- cells, suggesting that both Survivin and p21 block apoptosis of progenitors and that Survivin-mediated antiapoptosis requires p21. In contrast to the p21-dependent antiapoptotic effects, Survivin increased the proportion of CFU-GM in S-phase in both p21+/+ and p21-/- cells. Furthermore, modulating Survivin expression increased polyploidy in c-kit+, Lin- cells, which was accentuated by p21 deficiency. These results suggest that the Survivin-p21 axis plays an important role in the proliferation of normal hematopoietic cells and that Survivin regulates apoptosis through a p21 WAF1/Cip1-dependent pathway but may control S-phase entry independent of p21.

Constitutive activation of serine/threonine kinase Akt causes uncontrolled cell-cycle progression in different cell types and in malignancy. To investigate how Akt activation modulates cell-cycle progression in vascular smooth muscle cells (SMCs) in vitro and in the intact animal, we inhibited Akt-dependent signaling by adenovirus-mediated transfection of a dominant-negative Akt mutant (AA-Akt). We observed reduced proliferation rate (P<0.01), DNA synthesis (P<0.01), and a significant arrest in G1/S exit (P<0.01) both in vitro in response to serum stimulation and in vivo after vascular injury. In vivo transfection of the balloon-injured vessel with AA-Akt reduced SMC proliferation, resulting in decreased neointima compared with control virus (P<0.01). These effects were at least in part modulated, both in vitro and in vivo, by increased p21Cip1 expression, as demonstrated by lack of effect of AA-Akt on cell proliferation in p21-/- mouse SMCs. In conclusion, this study demonstrates that Akt-dependent signaling enhances cell-cycle progression of nontransformed SMCs in vitro and in response to vascular injury in the intact animal. These results suggest a role for Akt signaling in modulating the response of normal tissues to stress and the response of the arterial wall to acute and possibly repetitive injuries that ultimately contribute to restenosis and atherosclerosis.

Despite the ability of myogenic progenitor cells (MPCs) to completely regenerate skeletal muscle following injury, little is known regarding the molecular program that regulates their proliferation and differentiation. Although mice lacking the cyclin-dependent kinase inhibitor p21 (p21-/-), develop normally, we report here that p21-/- MPCs display increased cell number and enhanced cell cycle progression compared with wild-type MPCs. Therefore, we hypothesized that p21-/- mice would demonstrate temporally enhanced regeneration following myotrauma. In response to cardiotoxin-induced injury, p21-/- skeletal muscle regeneration was significantly attenuated vs. regenerating wild-type muscle, contrary to the hypothesis. Regenerating p21-/- skeletal muscle displayed increased proliferative (PCNA positive) nuclei coincident with increased apoptotic nuclei (TUNEL positive) compared with wild-type muscle up to 3 wk after injury. Differentiation of p21-/- MPCs was markedly impaired and associated with increased apoptosis compared with wild-type MPCs, confirming that the impaired differentiation of the p21-/- MPCs was a cell autonomous event. No dysregulation of p27, p53, or p57 protein expression in differentiating p21-/- MPCs compared with wild-type MPCs was observed, suggesting that other compensatory mechanisms are responsible for the regeneration that ultimately occurs. On the basis of these findings, we propose that p21 is essential for the coordination of cell cycle exit and differentiation in the adult MPC population and that in the absence of p21, skeletal muscle regeneration is markedly impaired.

Targeting the ubiquitin-proteasome pathway has emerged as a promising approach for treating cancer. Bortezomib (VELCADE, formerly known as PS-341), a potent and reversible proteasome inhibitor, is being evaluated in clinical trials for treating multiple myeloma, and various other types of hematologic and solid tumors. Proteasome inhibitors are known to induce apoptosis in human cancer cells. Nevertheless, the mechanisms of apoptosis induced by proteasome inhibitors remain unclear. In this study, we investigated the role of p53 and its downstream targets in bortezomib-induced apoptosis in HCT116 human colon cancer cells. We demonstrated that bortezomib induced p53, and activated its downstream genes p21, PUMA and Bax in a p53-dependent fashion. However, apoptotic response to bortezomib was not affected by the deletion of p53. Surprisingly, we found that bortezomib-induced apoptosis was markedly enhanced in the p21-knockout cells, while significantly decreased in the BAX-knockout cells. Furthermore, in the cells deficient for both Bax and p21, apoptosis was restored to the level in the parental or the p53-deficient cells. The opposite effects of Bax and p21 were unrelated to the extent of proteasome inhibition, and were also observed in cells treated with different proteasome inhibitors. These results indicate that p53 downstream targets can collectively modulate apoptotic response to bortezomib and other proteasome inhibitors.

In mammalian cells, cyclin E-CDK2 complexes are activated in the late G1 phase of the cell cycle and are believed to have an essential role in promoting S-phase entry. We have targeted the murine genes CCNE1 and CCNE2, encoding cyclins E1 and E2. Whereas single knockout mice were viable, double knockout embryos died around midgestation. Strikingly, however, these embryos showed no overt defects in cell proliferation. Instead, we observed developmental phenotypes consistent with placental dysfunction. Mutant placentas had an overall normal structure, but the nuclei of trophoblast giant cells, which normally undergo endoreplication and reach elevated ploidies, showed a marked reduction in DNA content. We derived trophoblast stem cells from double knockout E3.5 blastocysts. These cells retained the ability to differentiate into giant cells in vitro, but were unable to undergo multiple rounds of DNA synthesis, demonstrating that the lack of endoreplication was a cell-autonomous defect. Thus, during embryonic development, the needs for E-type cyclins can be overcome in mitotic cycles but not in endoreplicating cells.

The mechanism of thermal radiosensitization is related to the inhibition of repair of radiation-induced DNA damage by heat. Due to the interaction of the gene p21/WAF1/CIP1 (now known as CDKN1A) with a variety of DNA repair proteins, its involvement in thermal radiosensitization was investigated. Two isogenetic human colorectal cancer cell lines with wild-type TP53 status were used. The 80S4 cell line was deficient in CDKN1A and the HCT116 cells were CDKN1A proficient. Both cell lines were significantly more sensitive to 44 degrees C than 42 degrees C heating (P < 0.01), and both cell lines expressed thermotolerance for heating times longer than about 2 h at the lower temperature. There were no significant differences in the X-radiation response of the two cell lines. Further, the two cell lines displayed similar cell survival levels after hyperthermia given before or after X radiation for both hyperthermia temperatures. Comparison of thermal enhancement ratios confirmed that there was no difference in the amount of thermal radiosensitization induced in the two cell lines. The induction and subsequent repair of DNA double-strand breaks, as measured by clamped homogeneous gel electrophoresis, was also the same in both cell lines. These findings strongly suggest that the gene CDKN1A does not play an important role in the expression of thermal radiosensitization.

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 G1 is a transcriptional target of the tumor suppressor p53, and its expression is increased after DNA damage. Recent data show that cyclin G1 can regulate the levels of p53 by a mechanism that involves dephosphorylation of Mdm2 by protein phosphatase 2A. To understand the biologic role of cyclin G1, we have generated cyclin G1-deficient mice. In agreement with previous results, we showed that these mice develop normally, and that proliferation and induction of cellular senescence in cyclin G1-deficient mouse embryo fibroblasts are indistinguishable from wild-type fibroblasts. However, we found that the p53 levels in the cyclin G1-deficient mice are 2-fold higher that in wild-type mice. Moreover, we showed that treatment of mice with the alkylating agent 1,4-bis[N,N'-di(ethylene)-phosphamide]piperazine (Dipin), followed by partial hepatectomy, decreased G1-S transition in cyclin G1-null hepatocytes as compared with wild type. Finally, we found a significant decrease in tumor incidence, mass, and malignancy in both male and female cyclin G1-null mice after treatment with the potent hepatocarcinogen N-diethylnitrosamine. Taken with recent published data, our results suggest that cyclin G1, together with Mdm2, constitute a part of a negative feedback system that attenuates the activity of p53. In conclusion, our data suggest that the decreased tumor susceptibility after loss of cyclin G1 function is caused by the increased tumor suppressor action of p53.

Malignant gliomas are distinguished from low-grade gliomas by their intense angiogenesis. In gliomas, p53 is the most frequently altered gene and is involved in the early phase of glioma development. In contrast, homozygous p16 gene deletion is more common in high-grade gliomas. In order to understand the mechanism by which gliomas become more angiogenic during the malignant transformation, we examined the relationship between thrombospondin-1, a negative regulator in angiogenesis, and these tumor suppressor genes in malignant gliomas. Human glioma cell line U-251 MG, which has mutated p53 and deleted p16, was transduced with recombinant replication-defective adenovirus vectors containing the cDNA of wild-type p53, p16, and p21. Only the induction of wild-type p53 enhanced expression of thrombospondin-1 mRNA and the protein in U-251 MG cells. Furthermore, thrombospondin-1 that was secreted in the culture medium was significantly increased (3.8-fold) as compared with that of the viral control 36 h after infection with Ad5CMV-p53. In the presence of wild-type p53 plasmid DNA, the promoter activity was increased 7.4-fold as compared with an empty expression vector control. These studies may suggest that mutation of p53 gene endows gliomas with an angiogenic phenotype by reducing thrombospondin-1 production as well as enhancing the angiogenesis inducers in the early phase of malignant progression.

In mammalian organs under normoxic conditions, O2 concentration ranges from 12% to <0.5%, with O2 approximately 14% in arterial blood and <10% in the myocardium. During mild hypoxia, myocardial O2 drops to approximately 1% to 3% or lower. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of PO2 results in perceived hyperoxia. We hypothesized that O2, even in marginal relative excess of the PO2 to which cardiac cells are adjusted, results in activation of specific signal transduction pathways that alter the phenotype and function of these cells. To test this hypothesis, cardiac fibroblasts (CFs) isolated from adult murine ventricle were cultured in 10% or 21% O2 (hyperoxia relative to the PO2 to which cells are adjusted in vivo) and were compared with those cultured in 3% O2 (mild hypoxia). Compared with cells cultured in 3% O2, cells that were cultured in 10% or 21% O2 demonstrated remarkable reversible G2/M arrest and a phenotype indicative of differentiation to myofibroblasts. These effects were independent of NADPH oxidase function. CFs exposed to high O2 exhibited higher levels of reactive oxygen species production. The molecular signature response to perceived hyperoxia included (1) induction of p21, cyclin D1, cyclin D2, cyclin G1, Fos-related antigen-2, and transforming growth factor-beta1, (2) lowered telomerase activity, and (3) activation of transforming growth factor-beta1 and p38 mitogen-activated protein kinase. CFs deficient in p21 were resistant to such O2 sensitivity. This study raises the vital broad-based issue of controlling ambient O2 during the culture of primary cells isolated from organs.

During the pathogenesis of rheumatoid arthritis (RA), the synovial fibroblasts increase in number and produce proinflammatory cytokines and matrix metalloproteinases (MMPs) that function to promote inflammation and joint destruction. Recent investigations have suggested that cell cycle activity and inflammation may be linked. However, little is known about the mechanisms responsible for the coordinate regulation of proliferation and the expression of proinflammatory molecules in RA synovial fibroblasts. Here, we demonstrate a 50 +/- 10% decrease in the expression of p21, a cell cycle inhibitor, in the synovial fibroblast population from RA compared with osteoarthritis (OA) synovial tissue. Moreover, p21 positivity in the synovial fibroblasts inversely correlates with medium synovial lining thickness (r = -0.76; p < 0.02). The expression of p21 is also reduced in isolated RA synovial fibroblasts compared with OA synovial fibroblasts. Adenovirus-mediated delivery of p21 (Ad-p21) arrests both RA and OA synovial fibroblasts in the G(0)/G(1) phase of the cell cycle without inducing cytotoxicity. However, the spontaneous production of IL-6 and MMP-1 is suppressed only in the Ad-p21-infected RA synovial fibroblasts, indicating a novel role for p21 in RA. Analyses of p21-deficient mouse synovial fibroblasts reveal a 100-fold increase in IL-6 protein and enhance IL-6 and MMP-3 mRNA. Restoration of p21, but not overexpression of Rb, which also induces G(0)/G(1) cell cycle arrest, decreases IL-6 synthesis in p21-null synovial fibroblasts. Furthermore, in RA synovial fibroblasts the ectopic expression of p21 reduces activation of the AP-1 transcription factor. Additionally, p21-null synovial fibroblasts display enhanced activation of AP-1 compared with wild-type synovial fibroblasts. These data suggest that alterations in p21 expression may activate AP-1 leading to enhanced proinflammatory cytokine and MMP production and development of autoimmune disease.

Events that contribute to tumor formation include mutations in the ras gene and loss or inactivation of cell cycle inhibitors such as p21(Cip1) and p27(Kip1). In our previous publication, we showed that mice expressing the MMTV/v-Ha-ras transgene developed tumors earlier and at higher multiplicities in the absence than in the presence of p21(Cip1). To further evaluate the combinatorial role of genetic alterations and loss of cell cycle inhibitors in tumorigenesis, we performed two companion studies. In the first study, wild type and p21(Cip1)-null mice were exposed to the chemical carcinogen, urethane. Similar to its effects in v-Ha-ras mice, loss of p21(Cip1) accelerated tumor onset and increased tumor multiplicity in urethane-treated mice. Lung tumors were the predominant tumor type in urethane-treated mice regardless of p21(Cip1) status. In the second study, tumor formation was monitored in v-Ha-ras mice expressing or lacking p27(Kip1). Unlike p21(Cip1), the absence of p27(Kip1) had no effect on the timing or multiplicity of tumor formation, which was largely restricted to mammary and salivary glands. However, once tumors appeared, they grew faster in p27(Kip1)-null mice than in p27(Kip1)-wild type mice. Increases in growth rate were particularly striking for salivary tumors in ras/p27(-/-) mice. Loss of p21(Cip1), on the other hand, had no effect on tumor growth rate in v-Ha-ras mice. Collectively, our data suggest that p21(Cip1) suppresses tumor formation elicited by multiple agents and that p21(Cip1) and p27(Kip1) suppress tumor formation in different ways.

During differentiation of skeletal myoblasts, MyoD promotes growth arrest through the induction of the cdk inhibitor p21 and the accumulation of hypophosphorylated RB protein. Myoblasts lacking RB function fail to accomplish full differentiation and undergo apoptosis. Here we show that exogenous MyoD induces apoptosis in several cell backgrounds sharing RB inactivation. This process is associated with increased levels of cell cycle-driving proteins and aberrant cell cycle progression. The inability of MyoD to induce apoptosis in a p21-null background, highlights a requirement of p21 in RB-regulated apoptosis during myogenesis. This pro-apoptotic function of p21 cannot be exerted by simple p21 over-expression, but requires the co-operation of MyoD. We also suggest that the essential aspect of p21 activity involved in such a process is related to its ability to induce the nuclear accumulation and aberrant activity of cyclin/cdk complexes. These results establish a novel link between MyoD, p21 and RB during myogenesis, providing new insights into the antagonism between muscle differentiation and loss of RB function.