A genetic system for tissue-specific inhibition of cell proliferation.

Cellular proliferation is a basic process during organ development, tissue homeostasis and disease progression. Likewise, after injury typically multiple cell lineages respond to various cues and proliferate to initiate repair and/or remodeling of the injured tissue. Unravelling the specific role of proliferation of one cell type and its lineage in the context of the whole organism during tissue regeneration and/or disease progression would provide valuable information on these processes. Here, we report a new genetic system that allows cell proliferation to be inhibited in a tissue-specific manner. We generated Cre- or Dre-inducible p21-GFP (ip21-GFP) transgenic mice that enable experimentally induced permanent cell cycle arrest of specific cell lineages of interest, while genetically marking these cells. This system allows for the inhibition of pathogenic cell proliferation. We found that cardiac fibroblast proliferation inhibition significantly reduced scar formation, and promoted neovascularization and cardiomyocyte survival. Additionally, we found that inhibition of one type of cell proliferation (namely, hepatocytes) induces the lineage conversion of another type cells (i.e. ductal cells) during tissue regeneration. These results validate the use of ip21-GFP mice as a new genetic tool for cell lineage-specific inhibition of cell proliferation in vivo.

Transient activation of p53 in G2 phase is sufficient to induce senescence.

DNA damage can result in a transient cell-cycle arrest or lead to permanent cell-cycle withdrawal. Here we show that the decision to irreversibly withdraw from the cell cycle is made within a few hours following damage in G2 cells. This permanent arrest is dependent on induction of p53 and p21, resulting in the nuclear retention of Cyclin B1. This rapid response is followed by the activation of the APC/C(Cdh1) (the anaphase-promoting complex/cyclosome and its coactivator Cdh1) several hours later. Inhibition of APC/C(Cdh1) activity fails to prevent cell-cycle withdrawal, whereas preventing nuclear retention of Cyclin B1 does allow cells to remain in cycle. Importantly, transient induction of p53 in G2 cells is sufficient to induce senescence. Taken together, these results indicate that a rapid and transient pulse of p53 in G2 can drive nuclear retention of Cyclin B1 as the first irreversible step in the onset of senescence.

Musashi2 promotes the progression of pancreatic cancer through a novel ISYNA1-p21/ZEB-1 pathway.

Our previous studies found overexpression of Musashi2 (MSI2) conduced to the progression and chemoresistance of pancreatic cancer (PC) by negative regulation of Numb and wild type p53 (wtp53). Now, we further investigated the novel signalling involved with MSI2 in PC. We identified inositol-3-phosphate synthase 1 (ISYNA1) as a novel tumour suppressor regulated by MSI2. High MSI2 and low ISYNA1 expression were prevalently observed in 91 PC tissues. ISYNA1 expression was negatively correlated with MSI2 expression, T stage, vascular permeation and poor prognosis in PC patients. What's more, patients expressed high MSI2 and low ISYNA1 level had a significant worse prognosis. And in wtp53 Capan-2 and SW1990 cells, ISYNA1 was downregulated by p53 silencing. ISYNA1 silencing promoted cell proliferation and cell cycle by inhibiting p21 and enhanced cell migration and invasion by upregulating ZEB-1. However, MSI2 silencing upregulated ISYNA1 and p21 but downregulated ZEB-1, which can be rescued by ISYNA1 silencing. Moreover, reduction of cell migration and invasion resulting from MSI2 silencing was significantly reversed by ISYNA1 silencing. In summary, MSI2 facilitates the development of PC through a novel ISYNA1-p21/ZEB-1 pathway, which provides new gene target therapy for PC.

MSCs attenuate hypoxia induced pulmonary hypertension by activating P53 and NF-kB signaling pathway through TNFα secretion.

Hypoxia could cause vascular smooth muscle hypertrophy, leading to high pulmonary circulation resistance, pulmonary artery (PA) pressure, even pulmonary arterial hypertension (PAH). Recent studies have demonstrated the ability of mesenchymal stem cell (MSC) to ameliorate PAH but the mechanism was controversial. In this study, we revealed that the growth rate of pulmonary artery smooth muscle cells (PASMCs) treated with hypoxia was significantly increased than normal and showed lower expression of potassium channels. However, cells co-cultured with MSC showed decreased proliferation capability and down-regulated expression of ion channel of PAMSCs. The protein array data showed that the changes of PAMSCs was substantially associated with a high level of tumor necrosis factor alpha (TNFα) secretion from MSC. We further demonstrated that TNFα rescued the cell behavior of PAMSCs through activating the expression of P53 and NF-kB and inducing cell cycle arrest by P21/CDK2/CDK4 downregulation. These findings suggested that MSCs could attenuate abnormal function of PAMSCs by TNFα secretion, which was more or less associated with the beneficial effects of MSC on improving PAH.

DNA damage signaling triggers degradation of histone methyltransferases through APC/C(Cdh1) in senescent cells.

Both the DNA damage response (DDR) and epigenetic mechanisms play key roles in the implementation of senescent phenotypes, but very little is known about how these two mechanisms are integrated to establish senescence-associated gene expression. Here we show that, in senescent cells, the DDR induces proteasomal degradation of G9a and GLP, major histone H3K9 mono- and dimethyltransferases, through Cdc14B- and p21(Waf1/Cip1)-dependent activation of APC/C(Cdh1) ubiquitin ligase, thereby causing a global decrease in H3K9 dimethylation, an epigenetic mark for euchromatic gene silencing. Interestingly, induction of IL-6 and IL-8, major players of the senescence-associated secretory phenotype (SASP), correlated with a decline of H3K9 dimethylation around the respective gene promoters and knockdown of Cdh1 abolished IL-6/IL-8 expression in senescent cells, suggesting that the APC/C(Cdh1)-G9a/GLP axis plays crucial roles in aspects of senescent phenotype. These findings establish a role for APC/C(Cdh1) and reveal how the DDR integrates with epigenetic processes to induce senescence-associated gene expression.

HIV-1 Vpr and p21 restrict LINE-1 mobility.

Long interspersed element-1 (LINE-1, L1) composes ∼17% of the human genome. However, genetic interactions between L1 and human immunodeficiency virus type 1 (HIV-1) remain poorly understood. In this study, we found that HIV-1 suppresses L1 retrotransposition. Notably, HIV-1 Vpr strongly inhibited retrotransposition without inhibiting L1 promoter activity. Since Vpr is known to regulate host cell cycle, we examined the possibility whether Vpr suppresses L1 retrotransposition in a cell cycle dependent manner. We showed that the inhibitory effect of a mutant Vpr (H71R), which is unable to arrest the cell cycle, was significantly relieved compared with that of wild-type Vpr, suggesting that Vpr suppresses L1 mobility in a cell cycle dependent manner. Furthermore, a host cell cycle regulator p21Waf1 strongly suppressed L1 retrotransposition. The N-terminal kinase inhibitory domain (KID) of p21 was required for this inhibitory effect. Another KID-containing host cell cycle regulator p27Kip1 also strongly suppressed L1 retrotransposition. We showed that Vpr and p21 coimmunoprecipitated with L1 ORF2p and they suppressed the L1 reverse transcriptase activity in LEAP assay, suggesting that Vpr and p21 inhibit ORF2p-mediated reverse transcription. Altogether, our results suggest that viral and host cell cycle regulatory machinery limit L1 mobility in cultured cells.

Tpbpa-Cre-mediated deletion of TFAP2C leads to deregulation of Cdkn1a, Akt1 and the ERK pathway, causing placental growth arrest.

Loss of TFAP2C in mouse leads to developmental defects in the extra-embryonic compartment with lethality at embryonic day (E)7.5. To investigate the requirement of TFAP2C in later placental development, deletion of TFAP2C was induced throughout extra-embryonic ectoderm at E6.5, leading to severe placental abnormalities caused by reduced trophoblast population and resulting in embryonic retardation by E8.5. Deletion of TFAP2C in TPBPA(+) progenitors at E8.5 results in growth arrest of the junctional zone. TFAP2C regulates its target genes Cdkn1a (previously p21) and Dusp6, which are involved in repression of MAPK signaling. Loss of TFAP2C reduces activation of ERK1/2 in the placenta. Downregulation of Akt1 and reduced activation of phosphorylated AKT in the mutant placenta are accompanied by impaired glycogen synthesis. Loss of TFAP2C led to upregulation of imprinted gene H19 and downregulation of Slc38a4 and Ascl2. The placental insufficiency post E16.5 causes fetal growth restriction, with 19% lighter mutant pups. Knockdown of TFAP2C in human trophoblast choriocarcinoma JAr cells inhibited MAPK and AKT signaling. Thus, we present a model where TFAP2C in trophoblasts controls proliferation by repressing Cdkn1a and activating the MAPK pathway, further supporting differentiation of glycogen cells by activating the AKT pathway.

Dexamethasone induces primary amnion epithelial cell senescence through telomere-P21 associated pathway†.

Dexamethasone (Dex), a corticosteroid hormone, is used during the perinatal period to help fetal lung and other organ development. Conversely, Dex-induced cell proliferation has been associated with accelerated aging. Using primary amnion epithelial cells (AECs) from term, not in labor, fetal membranes, we tested the effects of Dex on cell proliferation, senescence, and inflammation. Primary AECs treated with Dex (100 and 200 nM) for 48 h were tested for cell viability (crystal violet dye exclusion), cell cycle progression and/or type of cell death (flow cytometry), expression patterns of steroid receptors (glucocorticoid receptor, progesterone receptor membrane component 1&2), inflammatory mediators (IL-6 and IL-8), and telomere length (quantitative RT-PCR). Mechanistic mediators of senescence (p38MAPK and p21) were determined by western blot analysis. Dex treatment did not induce AEC proliferation, cell cycle, influence viability, or morphology. However, Dex caused dependent telomere length reduction and p38MAPK-independent but p21-dependent (confirmed by treatment with p21 inhibitor UC2288). Senescence was not associated with an increase in inflammatory mediators, which is often associated with senescence. Co-treatment with RU486 produced DNA damage, cell cycle arrest, and cellular necrosis with an increase in inflammatory mediators. The effect of Dex was devoid of changes to steroid receptors, whereas RU486 increased GR expression. Dex treatment of AECs produced nonreplicative and noninflammatory senescence. Extensive use of Dex during the perinatal period may lead to cellular senescence, contributing to cellular aging associated pathologies during the perinatal and neonatal periods.

Upregulation of microRNA-17-5p contributes to hypoxia-induced proliferation in human pulmonary artery smooth muscle cells through modulation of p21 and PTEN.

BACKGROUND: Pulmonary arterial smooth muscle cell (PASMC) proliferation in response to hypoxia plays an important role in the vascular remodelling that occurs in hypoxic pulmonary hypertension. MicroRNAs (miRs) are emerging as important regulators in the progression of pulmonary hypertension. In this study, we investigated whether the expression of miR-17-5p is modulated by hypoxia and is involved in the hypoxia-induced proliferation of PASMCs. METHODS: Human PASMCs were cultured under hypoxic conditions. miR-17-5p expression was determined by real-time RT-PCR. A BrdU incorporation assay and time-lapse recording were utilized to determine cell proliferation and migration. RESULTS: PASMC proliferation was increased by moderate hypoxia (3% oxygen) but was reduced by severe hypoxia (0.1% oxygen) after 48 h. Moderate hypoxia induced miR-17-5p expression. Overexpression of miR-17-5p by transfection with miR-17-5p enhanced cell proliferation and migration in normoxia, whereas knockdown of miR-17-5p with anti-miR-17-5p inhibitors significantly reduced cell proliferation and migration. The expression of miR-17-5p target genes, specifically phosphatase and tensin homologue (PTEN) and cyclin-dependent kinase inhibitor 1 (p21WAF1/Cip1, p21), was reduced under moderate hypoxia in PASMCs. Under normoxia, overexpression of miR-17-5p in PASMCs reduced the expression of PTEN and p21. CONCLUSION: Our data indicate that miR-17-5p might play a significant role in hypoxia-induced pulmonary vascular smooth muscle cell proliferation by regulating multiple gene targets, including PTEN and p21, and that miR-17-5p could be a novel therapeutic target for the management of hypoxia-induced PH.

New origin firing is inhibited by APC/CCdh1 activation in S-phase after severe replication stress.

Defects in DNA replication and repair are known to promote genomic instability, a hallmark of cancer cells. Thus, eukaryotic cells have developed complex mechanisms to ensure accurate duplication of their genomes. While DNA damage response has been extensively studied in tumour cells, the pathways implicated in the response to replication stress are less well understood especially in non-transformed cells. Here we show that in non-transformed cells, APC/C(Cdh1) is activated upon severe replication stress. Activation of APC/C(Cdh1) prevents new origin firing and induces permanent arrest in S-phase. Moreover, Rad51-mediated homologous recombination is also impaired under these conditions. APC/C(Cdh1) activation in S-phase occurs after replication forks have been processed into double strand breaks. Remarkably, this activation, which correlates with decreased Emi1 levels, is not prevented by ATR/ATM inhibition, but it is abrogated in cells depleted of p53 or p21. Importantly, we found that the lack of APC/C(Cdh1) activity correlated with an increase in genomic instability. Taken together, our results define a new APC/C(Cdh1) function that prevents cell cycle resumption after prolonged replication stress by inhibiting origin firing, which may act as an additional mechanism in safeguarding genome integrity.

Direct interaction between Nrf2 and p21(Cip1/WAF1) upregulates the Nrf2-mediated antioxidant response.

In response to oxidative stress, Nrf2 and p21(Cip1/WAF1) are both upregulated to protect cells from oxidative damage. Nrf2 is constantly ubiquitinated by a Keap1 dimer that interacts with a weak-binding (29)DLG motif and a strong-binding (79)ETGE motif in Nrf2, resulting in degradation of Nrf2. Modification of the redox-sensitive cysteine residues on Keap1 disrupts the Keap1-(29)DLG binding, leading to diminished Nrf2 ubiquitination and activation of the antioxidant response. However, the underlying mechanism by which p21 protects cells from oxidative damage remains unclear. Here we present molecular and genetic evidence suggesting that the antioxidant function of p21 is mediated through activation of Nrf2 by stabilizing the Nrf2 protein. The (154)KRR motif in p21 directly interacts with the (29)DLG and (79)ETGE motifs in Nrf2 and thus competes with Keap1 for Nrf2 binding, compromising ubiquitination of Nrf2. Furthermore, the physiological significance of our findings was demonstrated in vivo using p21-deficient mice.

The guardian recruits cops: the p53-p21 axis delegates prosurvival duties to the Keap1-Nrf2 stress pathway.

In this issue of Molecular Cell, Zhang and colleagues (Chen et al., 2009) report their identification of p21 as a positive regulator of Nrf2, which extends p53 prosurvival functions through recruitment of a master stress-response regulator.

p21-/- mice exhibit enhanced bone regeneration after injury.

BACKGROUND: p21(WAF1/CIP1/SDI1), a cyclin dependent kinase inhibitor has been shown to influence cell proliferation, differentiation and apoptosis; but more recently, p21 has been implicated in tissue repair. Studies on p21(-/-) knockout mice have demonstrated results that vary from complete regeneration and healing of tissue to attenuated healing. There have however been no studies that have evaluated the role of p21 inhibition in bone healing and remodeling. METHODS: The current study employs a burr-hole fracture model to investigate bone regeneration subsequent to an injury in a p21-/- mouse model. p21-/- and C57BL/6 mice were subjected to a burr-hole fracture on their proximal tibia, and their bony parameters were measured over 4 weeks via in vivo µCT scanning. RESULTS: p21-/- mice present with enhanced healing from week 1 through week 4. Differences in bone formation and resorption potential between the two mouse models are assessed via quantitative and functional assays. While the µCT analysis indicates that p21-/- mice have enhanced bone healing capabilities, it appears that the differences observed may not be due to the function of osteoblasts or osteoclasts. Furthermore, no differences were observed in the differentiation of progenitor cells (mesenchymal or monocytic) into osteoblasts or osteoclasts respectively. CONCLUSIONS: Therefore, it remains unknown how p21 is regulating enhanced fracture repair and further studies are required to determine which cell type(s) are responsible for this regenerative phenotype.

AMPKα1 deficiency promotes cellular proliferation and DNA damage via p21 reduction in mouse embryonic fibroblasts.

Emerging evidence suggests that activation of adenosine monophosphate-activated protein kinase (AMPK), an energy gauge and redox sensor, controls the cell cycle and protects against DNA damage. However, the molecular mechanisms by which AMPKα isoform regulates DNA damage remain largely unknown. The aim of this study was to determine if AMPKα deletion contributes to cellular hyperproliferation by reducing p21(WAF1/Cip1) (p21) expression thereby leading to accumulated DNA damage. The markers for DNA damage, cell cycle proteins, and apoptosis were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J), AMPKα1, or AMPKα2 homozygous deficient (AMPKα1(-/-), AMPKα2(-/-)) mice by Western blot, flow cytometry, and cellular immunofluorescence staining. Deletion of AMPKα1, the predominant AMPKα isoform, but not AMPKα2 in immortalized MEFs led to spontaneous DNA double-strand breaks (DSB) which corresponded to repair protein p53-binding protein 1 (53BP1) foci formation and subsequent apoptosis. Furthermore, AMPKα1 localizes to chromatin and AMPKα1 deletion down-regulates cyclin-dependent kinase inhibitor, p21, an important protein that plays a role in decreasing the incidence of spontaneous DSB via inhibition of cell proliferation. In addition, AMPKα1 null cells exhibited enhanced cell proliferation. Finally, p21 overexpression partially blocked the cellular hyperproliferation of AMPKα1-deleted MEFs via the inhibition of cyclin-dependent kinase 2 (CDK2). Taken together, our results suggest that AMPKα1 plays a fundamental role in controlling the cell cycle thereby affecting DNA damage and cellular apoptosis.

Retinoic acid-related orphan receptor alpha reprograms glucose metabolism in glutamine-deficient hepatoma cells.

UNLABELLED: The metabolism of glutamine and glucose is recognized as a promising therapeutic target for the treatment of cancer; however, targeted molecules that mediate glutamine and glucose metabolism in cancer cells have not been addressed. Here, we show that restricting the supply of glutamine in hepatoma cells, including HepG2 and Hep3B cells, markedly increased the expression of retinoic acid-related orphan receptor alpha (RORα). Up-regulation of RORα in glutamine-deficient hepatoma cells resulted from an increase in the level of cellular reactive oxygen species and in the nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate reduced (NADP+ /NADPH) ratio, which was consistent with a reduction in the glutathione/glutathione disulfide (GSH/GSSG) ratio. Adenovirus (Ad)-mediated overexpression of RORα (Ad-RORα) or treatment with the RORα activator, SR1078, reduced aerobic glycolysis and down-regulated biosynthetic pathways in hepatoma cells. Ad-RORα and SR1078 reduced the expression of pyruvate dehydrogenase kinase 2 (PDK2) and inhibited the phosphorylation of pyruvate dehydrogenase and subsequently shifted pyruvate to complete oxidation. The RORα-mediated decrease in PDK2 levels was caused by up-regulation of p21, rather than p53. Furthermore, RORα inhibited hepatoma growth both in vitro and in a xenograft model in vivo. We also found that suppression of PDK2 inhibited hepatoma growth in a xenograft model. These findings mimic the altered glucose utilization and hepatoma growth caused by glutamine deprivation. Finally, tumor tissue from 187 hepatocellular carcinoma patients expressed lower levels of RORα than adjacent nontumor tissue, supporting a potential beneficial effect of RORα activation in the treatment of liver cancer. CONCLUSION: RORα mediates reprogramming of glucose metabolism in hepatoma cells in response to glutamine deficiency. The relationships established here between glutamine metabolism, RORα expression and signaling, and aerobic glycolysis have implications for therapeutic targeting of liver cancer metabolism.

Lenalidomide inhibits the proliferation of CLL cells via a cereblon/p21(WAF1/Cip1)-dependent mechanism independent of functional p53.

Lenalidomide has demonstrated clinical activity in patients with chronic lymphocytic leukemia (CLL), even though it is not cytotoxic for primary CLL cells in vitro. We examined the direct effect of lenalidomide on CLL-cell proliferation induced by CD154-expressing accessory cells in media containing interleukin-4 and -10. Treatment with lenalidomide significantly inhibited CLL-cell proliferation, an effect that was associated with the p53-independent upregulation of the cyclin-dependent kinase inhibitor, p21(WAF1/Cip1) (p21). Silencing p21 with small interfering RNA impaired the capacity of lenalidomide to inhibit CLL-cell proliferation. Silencing cereblon, a known molecular target of lenalidomide, impaired the capacity of lenalidomide to induce expression of p21, inhibit CD154-induced CLL-cell proliferation, or enhance the degradation of Ikaros family zinc finger proteins 1 and 3. We isolated CLL cells from the blood of patients before and after short-term treatment with low-dose lenalidomide (5 mg per day) and found the leukemia cells were also induced to express p21 in vivo. These results indicate that lenalidomide can directly inhibit proliferation of CLL cells in a cereblon/p21-dependent but p53-independent manner, at concentrations achievable in vivo, potentially contributing to the capacity of this drug to inhibit disease-progression in patients with CLL.

Roles of CDKN1A gene polymorphisms (rs1801270 and rs1059234) in the development of cervical neoplasia.

The CDKN1A gene product is a p53 downstream effector, which participates in cell differentiation, development process, repair, apoptosis, senescence, migration, and tumorigenesis. The objective of our study was investigated the importance of two polymorphisms in the CDKN1A gene, rs1801270 (31C>A) and rs1059234 (70C>T), for the development of cervical lesions in a Southeastern Brazilian population (283 cases, stratified by lesion severity, and 189 controls). CDKN1A genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and/or DNA sequencing. CDKN1A 31A allele presents a genetic pattern of protection for the development of high-grade cervical lesions (CC vs CA genotype: OR = 0.60; 95 % CI = 0.38-0.95; p = 0.029; CA+AA vs CC genotype: OR = 0.60; 95 % CI = 0.39-0.93; p = 0.021). Allele distributions of the CDKN1A 70C>T polymorphism were also different between the two study groups, with the CDKN1A 70T allele being less prevalent among cases. Moreover, the double heterozygote genotype combination 31CA-70CT decreases the chance of developing high-grade squamous intraepithelial lesion (HSIL) and cancer (OR = 0.55; 95 % CI = 0.32-0.93; p = 0.034) by 50 %, representing a protective factor against the development of more severe cervical lesions.

Polycomb protein SCML2 regulates the cell cycle by binding and modulating CDK/CYCLIN/p21 complexes.

Polycomb group (PcG) proteins are transcriptional repressors of genes involved in development and differentiation, and also maintain repression of key genes involved in the cell cycle, indirectly regulating cell proliferation. The human SCML2 gene, a mammalian homologue of the Drosophila PcG protein SCM, encodes two protein isoforms: SCML2A that is bound to chromatin and SCML2B that is predominantly nucleoplasmic. Here, we purified SCML2B and found that it forms a stable complex with CDK/CYCLIN/p21 and p27, enhancing the inhibitory effect of p21/p27. SCML2B participates in the G1/S checkpoint by stabilizing p21 and favoring its interaction with CDK2/CYCE, resulting in decreased kinase activity and inhibited progression through G1. In turn, CDK/CYCLIN complexes phosphorylate SCML2, and the interaction of SCML2B with CDK2 is regulated through the cell cycle. These findings highlight a direct crosstalk between the Polycomb system of cellular memory and the cell-cycle machinery in mammals.

p21(WAF1) (/Cip1) limits senescence and acinar-to-ductal metaplasia formation during pancreatitis.

Trans-differentiation of pancreatic acinar cells into ductal-like lesions, a process defined as acinar-to-ductal metaplasia (ADM), is observed in the course of organ regeneration following pancreatitis. In addition, ADM is found in association with pre-malignant PanIN lesions and correlates with an increased risk of pancreatic adenocarcinoma (PDAC). Human PDAC samples show down-regulation of p21(WAF1) (/Cip1) , a key regulator of cell cycle and cell differentiation. Here we investigated whether p21 down-regulation is implicated in controlling the early events of acinar cell trans-differentiation and ADM formation. p21-mediated regulation of ADM formation and regression was analysed in vivo during the course of cerulein-induced pancreatitis, using wild-type (WT) and p21-deficient (p21(-/-) ) mice. Biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks of the disease and during a recovery phase. We found that p21 was strongly up-regulated in WT acinar cells during pancreatitis, while it was absent in ADM areas, suggesting that p21 down-regulation is associated with ADM formation. In support of this hypothesis, p21(-/-) mice showed a significant increase in number and size of metaplasia. In addition, p21 over-expression in acinar cells reduced ADM formation in vitro, suggesting that the protein regulates the metaplastic transition in a cell-autonomous manner. p21(-/-) mice displayed increased expression and relocalization of ß-catenin both during pancreatitis and in the subsequent recovery phase. Finally, loss of p21 was accompanied by increased DNA damage and development of senescence. Our findings are consistent with a gate-keeper role of p21 in acinar cells to limit senescence activation and ADM formation during pancreatic regeneration.

p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells.

Apart from regulating stem cell self-renewal, embryonic development and proliferation, Bmi-1 has been recently reported to be critical in the maintenance of genome integrity. In searching for novel mechanisms underlying the anticlastogenic function of Bmi-1, we observed, for the first time, that Bmi-1 positively regulates p21 expression. We extended the finding that Bmi-1 deficiency induced chromosome breaks in multiple cancer cell models. Interestingly, we further demonstrated that knockdown of cyclin E or ectopic overexpression of p21 rescued Bmi-1 deficiency-induced chromosome breaks. We therefore conclude that p21/cyclin E pathway is crucial in modulating the anticlastogenic function of Bmi-1. As it is well established that the overexpression of cyclin E potently induces genome instability and p21 suppresses the function of cyclin E, the novel and important implication from our findings is that Bmi-1 plays an important role in limiting genomic instability in cylin E-overexpressing cancer cells by positive regulation of p21.