Positive Cell Cycle Regulators Research Articles

CRLF2 over-Expression Is a Poor Prognostic Marker in Children with High Risk T-Cell Acute Lymphoblastic Leukemia

Introduction: Although risk-adapted therapy improved the prognosis of pediatric T-ALL in the last decades, patients with T-ALL still have a worse outcome compared to B Cell Precursor (BCP)-ALL, and therefore they would benefit from the identification of new prognostic markers for a better treatment stratification and novel strategies to complement current chemotherapy regimens.Among newly reported genomic abnormalities, a subset of BCP-ALL patients is characterized by the over-expression of the Cytokine Receptor-like Factor 2 (CRLF2) gene, due to either an intra-chromosomal deletion causing the P2RY8-CRLF2 fusion or the IGH@-CRLF2 translocation. These two CRLF2 rearrangements were shown to correlate with poor outcome in BCP-ALL patients. In T-ALL, alterations of CRLF2 were not reported yet, but recently, mutations in its partner IL7Rα have been identified in about 10% of T-ALL patients.Aim: To estimate the incidence of CRLF2 aberrations and their prognostic value in pediatric T-ALL.Methods: We analyzed CRLF2 gene expression in 120 T-ALL patients, consecutively enrolled in the AIEOP-BFM ALL2000 study in Italian centers (AIEOP) from September 2000 to July 2005, and, as a validation cohort, in 92 consecutive patients treated with the same protocol in German centers (BFM-G), from July 1999 to December 2004. CRLF2 transcript levels were analyzed by RQ-PCR. Relative gene expression of CRLF2 was quantified by the 2-DDCt method. The DDCt for AIEOP and BFM-G samples was referred to the median DCt of their respective cohort.Results: An heterogeneous expression of CRLF2 was observed among AIEOP T-ALL patients (range: 0.06 to 82 fold change). Seventeen patients (14.2%) presented an expression 5 times higher than the median ('CRLF2-high’). Interestingly, none of the CRLF2-high cases resulted to be positive for P2RY8-CRLF2 fusion, 1/5 was positive for the IGH@ translocation and 1/7 showed a supernumerary X chromosome. JAK2 and CRLF2 mutations were absent in all 120 cases, while IL7R mutations were detected in 5/107 patients (4.7%), but unexpectedly they were not associated to CRLF2 over-expression.CRLF2-high patients had a significantly inferior 5-y EFS (41.2%±11.9 vs. 68.9%±4.6, p=0.006) and an increased, although not statistically significant, cumulative incidence of relapse (CIR) compared to CRLF2-low patients (41.2%±11.9 vs. 26.3%±4.3, p=0.17).The prognostic value of CRLF2 over-expression was confirmed in the BFM-G cohort (5-y EFS in 12 CRLF2-high patients was 50.0%±14.4 vs 83.8%±4.1, p-=0.008; CIR: 33.3%±13.6 vs. 11.3%±3.5, p= 0.06).Interestingly, CRLF2-high patients were more frequently allocated to the high risk (HR) T-ALL subgroup (20.9% in HR vs.8.3% in no-HR in the two cohorts analyzed together). In this subgroup, CRLF2 over-expression was significantly associated to a poor prognosis (5-y EFS: 31.6%±10.7 vs. 62.5%±5.7, p-value=0.008; CIR: 47.4%±11.5 vs. 29.2%±5.4, p=0.14).When analyzed according to prednisone (PDN) response, 17/28 (61%) were prednisone poor responders (PPR), and in the 'PPR-only’ subgroup (non-HR by other features) 4/9 (44%) CRLF2-high patients relapsed versus 4/36 (11%) CRLF2-low.Cox model analysis adjusted by risk group showed that CRLF2-high expression had a relevant prognostic impact with a 2-fold increased risk of relapse (Hazard ratio 2.12; 95% CI 1.07-4.21; p=0.03).The mechanisms responsible for CRLF2 over-expression in T-ALL and its contribution to the pathogenesis of the disease is still being investigated. Notably, gene set enrichment analysis (GSEA) showed an inverse correlation between the expression of CRLF2 and positive cell cycle regulators, thus suggesting that CRLF2-high blasts have a low proliferating activity and may therefore be less sensitive to conventional chemotherapy.Conclusions: CRLF2 over-expression is a poor prognostic marker not only in BCP-ALL patients, but also in T-ALL, identifying a subset of HR T-ALL patients with extremely severe outcome. Specifically, this marker would allow identifying T-ALL patients that could benefit from alternative therapy, potentially targetting the CRLF2 pathway. DisclosuresNo relevant conflicts of interest to declare.

Effect of SMAD7 gene overexpression on TGF-β1-induced profibrotic responses in fibroblasts derived from Peyronie's plaque.

Transforming growth factor-β1 (TGF-β1) has been identified as one of the most important fibrogenic cytokines associated with Peyronie's disease (PD). The mothers against decapentaplegic homolog 7 (SMAD7) is an inhibitory Smad protein that blocks TGF-β signaling pathway. The aim of this study was to examine the anti-fibrotic effect of the SMAD7 gene in primary fibroblasts derived from human PD plaques. PD fibroblasts were pretreated with the SMAD7 gene and then stimulated with TGF-β1. Treated fibroblasts were used for Western blotting, fluorescent immunocytochemistry, hydroxyproline determination, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. Overexpression of the SMAD7 gene inhibited TGF-β1-induced phosphorylation and nuclear translocation of SMAD2 and SMAD3, transdifferentiation of fibroblasts into myofibroblasts, and quashed TGF-β1-induced production of extracellular matrix protein and hydroxyproline. Overexpression of the SMAD7 gene decreased the expression of cyclin D1 (a positive cell cycle regulator) and induced the expression of poly (ADP-ribose) polymerase 1, which is known to terminate Smad-mediated transcription, in PD fibroblasts. These findings suggest that the blocking of the TGF-β pathway by use of SMAD7 may be a promising therapeutic strategy for the treatment of PD.

Connective tissue growth factor modulates adult β-cell maturity and proliferation to promote β-cell regeneration in mice.

Stimulation of endogenous β-cell expansion could facilitate regeneration in patients with diabetes. In mice, connective tissue growth factor (CTGF) is expressed in embryonic β-cells and in adult β-cells during periods of expansion. We discovered that in embryos CTGF is necessary for β-cell proliferation, and increased CTGF in β-cells promotes proliferation of immature (MafA−) insulin-positive cells. CTGF overexpression, under nonstimulatory conditions, does not increase adult β-cell proliferation. In this study, we tested the ability of CTGF to promote β-cell proliferation and regeneration after partial β-cell destruction. β-Cell mass reaches 50% recovery after 4 weeks of CTGF treatment, primarily via increased β-cell proliferation, which is enhanced as early as 2 days of treatment. CTGF treatment increases the number of immature β-cells but promotes proliferation of both mature and immature β-cells. A shortened β-cell replication refractory period is also observed. CTGF treatment upregulates positive cell-cycle regulators and factors involved in β-cell proliferation, including hepatocyte growth factor, serotonin synthesis, and integrin β1. Ex vivo treatment of whole islets with recombinant human CTGF induces β-cell replication and gene expression changes consistent with those observed in vivo, demonstrating that CTGF acts directly on islets to promote β-cell replication. Thus, CTGF can induce replication of adult mouse β-cells given a permissive microenvironment.

Molecular mechanisms associated with PTHrP-induced proliferation of colon cancer cells.

Parathyroid Hormone-related Protein (PTHrP) is normally produced in many tissues and is recognized for its endocrine, paracrine, autocrine and intracrine modes of action. PTHrP is also implicated in different types of cancer and its expression correlates with the severity of colon carcinoma. Using the human colon cell line Caco-2 we recently obtained evidence that PTHrP, through a paracrine pathway, exerts a protective effect under apoptotic conditions. However, if exogenous PTHrP is able or not to induce the proliferation of these intestinal tumor cells is not known. We found that PTHrP treatment increases the number of live Caco-2 cells. The hormone induces the phosphorylation and nuclear translocation of ERK 1/2, α p38 MAPK, and Akt, without affecting JNK phosphorylation. In addition, PTHrP-dependent ERK phosphorylation is reverted when PI3K activity was inhibited. Following MAPKs nuclear translocation, the transcription factors ATF-1 and CREB were activated in a biphasic manner. In addition PTHrP induces the translocation into the nucleus of β-catenin, protein that plays key role in maintaining the growth and proliferation of colorectal cancer, and increases the amount of both positive cell cycle regulators c-Myc and Cyclin D. Studies with ERK1/2, α p38 MAPK, and PI3K specific inhibitors showed that PTHrP regulates Caco-2 cell proliferation via these signaling pathways. In conclusion, the results obtained in this work expand our knowledge on the role of exogenous PTHrP in intestinal tumor cells and identify the signaling pathways that are involved in the mitogenic effect of the hormone on Caco-2 cells.

Silencing histone deacetylase 2 using small hairpin RNA induces regression of fibrotic plaque in a rat model of Peyronie's disease.

To examine the therapeutic effect of adenovirus encoding histone deacetylase 2 (HDAC2) small hairpin RNA (Ad-HDAC2 shRNA) in a rat model of Peyronie's disease (PD) and to determine the mechanisms by which HDAC2 knockdown ameliorates fibrotic responses in primary fibroblasts derived from human PD plaque. Rats were distributed into four groups (n = 6 per group): age-matched controls without treatment; rats in which PD has been induced (PD rats) without treatment; PD rats receiving a single injection of control adenovirus encoding scrambled small hairpin RNA (Ad-shRNA) (day 15; 1 × 10(8) pfu/0.1 mL phosphate-buffered saline [PBS]); and PD rats receiving a single injection of Ad-HDAC2 shRNA (day 15; 1 × 10(8) pfu/0.1 mL PBS) into the lesion. PD-like plaque was induced by repeated intratunical injections of 100 μL each of human fibrin and thrombin solutions on days 0 and 5. On day 30, the penis was harvested for histological examination. Fibroblasts isolated from human PD plaque were pretreated with HDAC2 small interfering (si)RNA (100 pmoL) and then stimulated with transforming growth factor (TGF)-β1 (10 ng/mL) to determine hydroxyproline levels, procollagen mRNA, apoptosis and protein expression of poly(ADP-ribose) polymerase 1 (PARP1) and cyclin D1. We observed that Ad-HDAC2 shRNA decreased inflammatory cell infiltration, reduced transnuclear expression of phospho-Smad3 and regressed fibrotic plaque of the tunica albuginea in PD rats in vivo. siRNA-mediated silencing of HDAC2 significantly decreased the TGF-β1-induced transdifferentiation of fibroblasts into myofibroblasts and collagen production, and induced apoptosis by downregulating the expression of PARP1, and decreased the expression of cyclin D1 (a positive cell-cycle regulator) in primary cultured fibroblasts derived from human PD plaque in vitro. Specific inhibition of HDAC2 with RNA interference may represent a novel targeted therapy for PD.

The role of androst-5-ene-3β,17β-diol (androstenediol) in cell proliferation in endometrium of women with polycystic ovary syndrome

Women with polycystic ovary syndrome (PCOS) show high prevalence of endometrial hyperplasia and adenocarcinoma. Endometrial proliferation is increased, evaluated by high levels of Ki67 (cell cycle marker) and low levels of p27 (negative regulator of cell cycle). Nevertheless, endometrial changes in cyclin D1 (positive regulator of cell cycle) in PCOS-women are not described. Androst-5-ene-3β,17β-diol (androstenediol), steroid with estrogenic activity present in endometria, could be related to increased endometrial cell proliferation. The objective of this study was to determine protein content of cyclin D1 and androstenediol levels in endometria from PCOS and control-women and to evaluate the possible mechanism favoring cell proliferation associated with hormonal characteristics of patients. Therefore, cyclin D1 protein content in PCOS-women and control-endometrial tissue were assessed by western blot and immunohistochemistry. The androstenediol levels were evaluated by ELISA. To further analyze the effect of steroids (androstenediol, 17β-estradiol, testosterone) in cell proliferation, levels of proteins cyclin D1, p27 and Ki67 were evaluated in an in vitro model of stromal endometrial cells T-HESC and St-T1b. An increase in cyclin D1 and androstenediol was observed in tissues from PCOS-women relative to control group (p<0.05). In the in vitro model, androstenediol exerted increase in cyclin D1 (p<0.05) and a decrease in p27 protein level (p<0.05), while Ki67 in St-T1b cells increased under this stimulus (p<0.05). Testosterone produces opposite effects in the levels of the above markers (p<0.05). Therefore, the hormonal imbalance associated with this syndrome could alter endometrial tissue homeostasis, promoting cell proliferation. Androstenediol is a molecule that could be involved by stimulating proliferation, whereas testosterone elicits a role of cell cycle repressor.

Altered microRNA expression profiles are involved in resistance to low-dose ionizing radiation in the absence of BMI1 in human dermal fibroblasts.

The polycomb group RING finger protein, B-cell‑specific moloney murine leukemia virus integration site1 (BMI1), has emerged as a key regulator of cell proliferation, cell cycle, cell immortalization, chemoresistance and radioresistance. Although the radioresistant effect of BMI1 has been thoroughly investigated, the effectiveness of this factor on low-dose radiation (LDR) resistance has not been explored. Here, we demonstrate that BMI1 is not critical for altering cell viability or cell growth in response to LDR, but BMI1 changes cellular gene expression profiles in response to LDR. Normal human dermal fibroblasts (NHDFs) stably expressing BMI1 short hairpin RNA (shRNA) did not exhibit changes in cell viability or cell cycle distribution assays following exposure to 0.1Gy of γ-radiation. However, microRNA (miRNA) microarrays revealed that a lack of BMI1 leads to changes in miRNA expression in response to LDR. Bioinformatics analyses demonstrated that predicted target genes of the altered miRNAs are functionally involved in both negative and positive regulation of cell growth, cell proliferation, cell cycle and apoptosis. Therefore, these results indicate that low radiosensitivity even in the absence of the radioresistant factor BMI1 is related with the altered miRNA expression profiles in NHDF.

Brucine inhibits the proliferation of human lung cancer cell line PC-9 via arresting cell cycle

背景与目的已有的研究表明：Cyclin D1和Cyclin E是细胞周期中重要的正性调控因子，其高表达与肿瘤的增殖密切相关。本研究旨在探讨马钱子碱（Brucine）对人肺癌细胞株PC-9增殖的影响，及其与Cyclin D1和Cyclin E表达的影响。方法将PC-9细胞分为4组：空白对照组、DMSO对照组（2‰）、150 μM Brucine组、300 μM Brucine组。CellTiter-Glo发光法、平板克隆形成实验观察该药对PC-9细胞增殖的影响，流式细胞仪检测细胞周期，qRT-PCR检测细胞周期相关基因Cyclin D1、Cyclin E mRNA的表达，Western blot检测细胞周期相关基因Cyclin D1、Cyclin E蛋白的表达。结果与对照组比较，CellTiter-Glo发光法、平板克隆形成实验结果显示：Brucine可以抑制人肺癌细胞株PC-9的增殖，并呈时间-剂量依赖性（P < 0.01）；流式结果显示对细胞周期的影响主要是阻滞PC-9细胞于G0/G1期；qRT-PCR结果显示Cyclin D1、Cyclin E mRNA的表达下调；Western blot结果显示Brucine使Cyclin D1、Cyclin E的表达降低。结论Brucine能明显抑制人肺癌细胞株PC-9的增殖，机制主要与其通过下调Cyclin D1、Cyclin E表达，进而阻滞细胞周期有关。

Rapamycin inhibits pre-B acute lymphoblastic leukemia cells by downregulating DNA and RNA polymerases

Rapamycin has been shown to inhibit the growth of leukemic cells via an unknown mechanism. In our current study, we show that rapamycin activates autophagy in pediatric t(1;19) pre-B acute lymphoblastic leukemia (pre-B ALL) cells and thereby inhibits proliferation and induces growth arrest in these cells. Rapamycin was found to downregulate an extensive array of positive cell cycle regulators, reduce the total DNA and RNA levels, and specifically downregulate the gene transcription of DNA pol δ1 and RNA pol II. Furthermore, we show that both rapamycin and starvation caused a downregulation of the DNA pol δ1 and RNA pol II proteins which was reversed by the autophagy inhibitor 3-MA. Consistent with the results of our autophagic flux analysis, confocal microscopy indicated that both rapamycin and starvation cause the colocalization of DNA pol δ1 and RNA pol II with GFP-LC3 at autophagosomes. This colocalization was blocked by the autophagy inhibitor bafilomycin A1 which inhibits the fusion between autophagosomes and lysosomes. These data suggest that rapamycin inhibits the growth of pediatric t(1;19) pre-B ALL cells through both transcriptional inhibition and autophagic degradation of DNA pol δ1 and RNA pol II.

A novel synthetic derivative of the natural product berbamine inhibits cell viability and induces apoptosis of human osteosarcoma cells, associated with activation of JNK/AP-1 signaling

Osteosarcoma is the most common primary bone tumor in children and adolescents. There is a critical need to find more potent drugs for patients with metastatic or recurrent disease. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plants. BBM and its derivatives have been shown to have antitumor effects in several cancers. Here, we report that a novel synthetic berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of G292, KHOS, and MG-63 human osteosarcoma cells. Induction of apoptosis in these tumor cells depends on activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). Since pan-caspase inhibitor (Z-VAD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) could block the cleavage of PARP, the apoptosis induced by BBMD3 is through intrinsic signaling pathway. BBMD3 increased phosphorylation of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), resulting in increase of phosphorylated c-Jun and total c-Fos, the major components of transcriptional factor AP-1. JNK inhibitor could partially suppress antitumor effect of BBMD3 on osteosarcoma cells. BBMD3 increased the production of reactive oxygen species (ROS) and ROS scavenger, N-acetylcysteine (NAC), could block the phosphorylation of JNK and c-Jun induced by BBMD3. BBMD3 increased the expression of the pro-apototic gene Bad, associated with apoptosis induction. Finally, BBMD3 also decreased the expression of cyclin D1 and D2, the positive cell cycle regulators, which is correlated with growth inhibition in osteosarcoma cells. Collectively, these findings indicate that BBMD3 is a potentially promising drug for the treatment of human osteosarcoma.

Small Molecule-Induced Mitochondrial Disruption Directs Prostate Cancer Inhibition via Unfolded Protein Response Signaling

We previously identified SMIP004 (N-(4-butyl-2-methyl-phenyl) acetamide) as a novel inducer of cancer-cell selective apoptosis of human prostate cancer cells. SMIP004 decreased the levels of positive cell cycle regulators, upregulated cyclin-dependent kinase inhibitors, and resulted in G1 arrest, inhibition of colony formation in soft agar, and cell death. However, the mechanism of SMIP004-induced cancer cell selective apoptosis remained unknown. Here, we used chemical genomic and proteomic profiling to unravel a SMIP004-induced pro-apoptotic pathway, which initiates with disruption of mitochondrial respiration leading to oxidative stress. This, in turn, activates two pathways, one eliciting cell cycle arrest by rapidly targeting cyclin D1 for proteasomal degradation and driving the transcriptional downregulation of the androgen receptor, and a second pathway that activates pro-apoptotic signaling through MAPK activation downstream of the unfolded protein response (UPR). SMIP004 potently inhibits the growth of prostate and breast cancer xenografts in mice. Our data suggest that SMIP004, by inducing mitochondrial ROS formation, targets specific sensitivities of prostate cancer cells to redox and bioenergetic imbalances that can be exploited in cancer therapy.

Ischemic Post-Conditioning Partially Reverses Cell Cycle Reactivity Following Ischemia/Reperfusion Injury: A Genome-Wide Survey

Neuronal cell cycle re-entry is pro-apoptotic. The neuroprotective effects and anti-apoptosis of ischemic postconditioning (IPostC) are well established but the underlying mechanism is still unknown. We explored this critical gap in the present study by genomic comparison of ischemic rat cortex following transient middle cerebral artery occlusion (tMCAO) alone and tMCAO+IPostC. The gene expression profiles of ipsilateral cortices were subjected to microarray analysis. RT-PCR, immunoblotting, and immunofluorescence were subsequently used to quantify or localize the cell proliferation marker proliferating cell nuclear antigen (PCNA), positive and negative cell cycle regulators, and related signaling molecules. Microarray analysis revealed that tMCAO-induced transcriptional changes in 40 cell cycle regulators were ameliorated by IPostC, suggesting that IPostC reversed neuronal cell cycle re-entry. IPostC reversed the rise in mRNA levels of positive cell cycle regulators ccnb1, cdk1, cdca2, cdca3, and cdca7. Elevations in cyclin D1 and neuronal cyclin A2 were similarly inhibited as well. tMCAO-induced phosphorylation of extracellular signal-regulated kinase (p-ERK), glycogen synthase kinase-3β (p-GSK-3β), and cAMP response element binding protein (p-CREB) were also all depressed by IPostC. Furthermore, p-ERK colocalized with neuronal cyclin A2. The present study demonstrates the potent inhibitory effect of IPostC treatment on tMCAO-induced cell-cycle reentry and on ERK/CREB and GSK- 3β/CREB signaling. Because neuronal cell cycle re-entry is pro-apoptotic, these findings lend insight into potential mechanisms underlying neuroprotection of IPostC.

Abstract WP117: Ischemic Post-conditioning Partially Reverses Cell Cycle Re-entry Following Transient Ischemia/Reperfusion Injury

Background and purpose —The neuroprotective effects of ischemic postconditioning (IPostC) are well established but the underlying mechanism is still unknown. We began to explore this critical gap in the field by genomic comparison of ischemic rat cortex following transient middle cerebral artery occlusion (tMCAO) alone or tMCAO+IPostC. Methods and Results —Microarray analysis revealed that tMCAO-induced transcriptional changes in 40 cell cycle regulators were ameliorated by IPostC, suggesting that IPostC reversed neuronal cell cycle re-entry. RT-PCR, immunoblotting, and immunofluorescence were subsequently used to quantify or localize the cell proliferation marker proliferating cell nuclear antigen (PCNA), positive and negative cell cycle regulators, and related signaling molecules. As expected, IPostC reversed the rise in mRNA levels of positive cell cycle regulators cdcb1, cdk1, cdca2, cadca3, and cdca7. Elevations in cyclin D1 and neuronal cyclin A2 were similarly inhibited. tMCAO-induced phosphorylation of extracellular signal-regulated kinase (p-ERK), glycogen synthase kinase-3β (p-GS3K3β), and cAMP response element binding protein (p-CREB) were also all depressed by IPostC. Furthermore, p-ERK colocalized with neuronal cyclin A2. Conclusions —The present study demonstrates the potent inhibitory effect of IPostC treatment on tMCAO-induced cell-cycle reentry and on ERK/CREB and GSK3β/CREB signaling. Because neuronal cell cycle re-entry is pro-apoptotic, these findings lend insight into potential mechanisms underlying neuroprotection with IPostC.

Differential Cellular and Molecular Effects of Butyrate and Trichostatin A on Vascular Smooth Muscle Cells

The histone deacetylase (HDAC) inhibitors, butyrate and trichostatin A (TSA), are epigenetic histone modifiers and proliferation inhibitors by downregulating cyclin D1, a positive cell cycle regulator, and upregulating p21Cip1 and INK family of proteins, negative cell cycle regulators. Our recent study indicated cyclin D1 upregulation in vascular smooth muscle cells (VSMC) that are proliferation-arrested by butyrate. Here we investigate whether cyclin D1 upregulation is a unique response of VSMC to butyrate or a general response to HDAC inhibitors (HDACi) by evaluating the effects of butyrate and TSA on VSMC. While butyrate and TSA inhibit VSMC proliferation via cytostatic and cytotoxic effects, respectively, they downregulate cdk4, cdk6, and cdk2, and upregulate cyclin D3, p21Cip1 and p15INK4B, and cause similar effects on key histone H3 posttranslational modifications. Conversely, cyclin D1 is upregulated by butyrate and inhibited by TSA. Assessment of glycogen synthase 3-dependent phosphorylation, subcellular localization and transcription of cyclin D1 indicates that differential effects of butyrate and TSA on cyclin D1 levels are linked to disparity in cyclin D1 gene expression. Disparity in butyrate- and TSA-induced cyclin D1 may influence transcriptional regulation of genes that are associated with changes in cellular morphology/cellular effects that these HDACi confer on VSMC, as a transcriptional modulator.

Role of D-type cyclins in heart development and disease

A defining feature of embryonic cardiomyocytes is their relatively high rates of proliferation. A gradual reduction in proliferative capacity throughout development culminates in permanent cell cycle exit by the vast majority of cardiomyocytes around the perinatal period. Accordingly, the adult heart has severely limited capacity for regeneration in response to injury or disease. The D-type cyclins (cyclin D1, D2, and D3) along with their catalytically active partners, the cyclin dependent kinases, are positive cell cycle regulators that play important roles in regulating proliferation of cardiomyocytes during normal heart development. While expression of D-type cyclins is generally low in the adult heart, expression levels are augmented in association with cardiac hypertrophy, but are uncoupled from myocyte cell division. Accordingly, re-activation of D-type cyclin expression in the adult heart has been implicated in pathophysiological processes via mechanisms distinct from those that drive proliferation during cardiac development. Growth factors and other exogenous agents regulate D-type cyclin production and activity in embryonic and adult cardiomyocytes. Understanding differences in the precise intracellular mediators downstream from these signalling molecules in embryonic versus adult cardiomyocytes could prove valuable for designing strategies to reactivate the cell cycle in cardiomyocytes in the setting of cardiovascular disease in the adult heart.

Notch signaling pathway regulates proliferation and differentiation of immortalized Müller cells under hypoxic conditions in vitro

Previous studies have indicated that Müller glia in chick and fish retinas can re-enter the cell cycle, express progenitor genes, and regenerate neurons via the Notch signaling pathway in response to retinal damage or growth factors. Here, we investigated the role of Notch signaling and the effect of hypoxia, as a means to induce retinal damage, on the proliferation of an immortalized Müller cell line (rMC-1 cells). Our data showed that rMC-1 cells expressed Müller glia and neural and retinal progenitor markers but did not express neuronal or retinal markers. Hypoxia increased rMC-1 cell proliferation by activating the positive cell-cycle regulators, cyclins A and D1, as well as the neural and retinal progenitor markers, Notch1, Hes1, nestin, Sox2, Msi1, Pax6, and NeuroD1. However, hypoxia did not significantly influence the expression of Müller glial markers GS, CRALBP, and cyclin D3 or the death of the rMC-1 cells. The increase in cell proliferation induced by hypoxia was greatly attenuated by blocking Notch signaling with the inhibitor DAPT, resulting in the reduced expression of positive cell-cycle regulators (cyclins A and D1) and neural and retinal progenitor markers (Notch1, Hes1, Sox2, Pax6, and NeuroD1). Blockade of the Notch signaling pathway by DAPT after hypoxia promoted the differentiation of rMC-1 cells to neurons, as demonstrated by the induction of neural marker (Tuj1), retinal amacrine (Syntaxin1), and retinal ganglion cell (Brn3b) markers, although the expression of the latter marker was low. Taken together, our data indicate that Notch signaling is required for proliferation under hypoxic conditions either by activating the positive cell-cycle regulators or by skewing their de-differentiation towards a neural progenitor lineage. These findings indicate that the Notch signaling pathway regulates hypoxia-induced proliferation and differentiation of Müller glia.

Abstract 2069: A novel berbamine derivative induces apoptosis of chemotherapy-resistant human osteosarcoma cells through activation of ROS/JNK signaling

Abstract Osteosarcoma is the most common primary bone tumor in children and adolescence. There is a critical need to find more potent drugs for patients with metastatic or recurrent disease. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plants. BBM and its derivatives have been shown to have anti-tumor effects in several cancers. Here, we report that a new berbamine derivative, BBMD3 (5 µM), induces apoptosis of human osteosarcoma cells, which are resistant or insensitive to conventional chemotherapy including cisplatin, doxorubicin and methotrexate. BBMD3 inhibited cell viability and induced apoptosis of G292, KHOS and MG-63 human osteosarcoma cells in a time- and dose-dependent manner. BBMD3 activated caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP). Caspase inhibitor (Z-VAD-FMK) could rescue tumor cells from the cytotoxic effects of BBMD3 on cell survival. Importantly, BBMD3 increased phosphorylation of c-Jun N-terminal kinase (JNK) in these tumor cells, resulting in activation of its substrates, c-Jun and c-Fos. Moreover, reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), blocked the phosphorylation of JNK and c-Jun induced by BBMD3. However, Z-VAD-FMK treatment did not inhibit the induction of phosphorylated JNK and c-Jun by BBMD3, suggesting that phosphorylation of JNK and c-Jun occurred before activation of caspases. BBMD3 increased the expression of the pro-apototic gene Bad, associated with apoptosis induction. Finally, BBMD3 also decreased the expression of cyclin D1, a positive cell cycle regulator, which is correlated with growth suppression in osteosarcoma cells. Collectively, these findings indicate that BBMD3 is a potentially promising drug for the treatment of chemotherapy-resistant osteosarcomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2069. doi:1538-7445.AM2012-2069

Abstract 2306: Overexpression of microRNA-223 regulates the ubiquitin ligase FBXW7 in esophageal squamous cell carcinoma

Abstract Background: F-box and WD repeat domain-containing 7 (FBXW7) is a cell cycle regulatory gene whose protein product ubiquitinates positive cell cycle regulators such as c-Myc, cyclin E and c-Jun, thereby acting as a tumor-suppressor gene. This study focused on microRNA-223 (miR-223), which is a candidate regulator of FBXW7 mRNA. The aim of this study was to clarify the clinical significance of miR-223 and FBXW7 in esophageal squamous cell carcinoma (ESCC) patients and to elucidate the mechanism by which FBXW7 is regulated by miR-223. Methods: The expression levels of miR-223 and the expression of FBXW7 protein was examined using 109 resected specimens to determine the clinicopathological significance. The expression levels of miR-223 were determined by qRT-PCR. The immunohistochemical studies for FBXW7 were performed on formalin-fixed paraffin-embedded surgical sections obtained from 109 patients with ESCC. We also investigated the role of miR-223 in the regulation of FBXW7 expression in ESCC cell lines in an in vitro analysis. Results: We found that the mean expression levels of miR-223 in cancerous tissue specimens were significantly higher than those in non-cancerous tissues (p &amp;lt;0.001). We divided the 109 ESCC patients into two groups according to the ratio of their cancer/normal tissue expression levels of miR-223, as α1.0 or &amp;lt;1.0 for the cancer/noncancerous tissues expression levels of miR-223. There were 74 cases (67.9%) in the high miR-223 group and 35 cases (32.1%) in the low miR-223 expression group. There were significant differences in gender (p = 0.008), tumor size (p = 0.042), and depth of tumor invasion (p = 0.030) between the groups. There was a significant inverse relationship between the expression levels of miR-223 and FBXW7 protein. High levels of miR-223 were associated with low FBXW7 expression (Pearson correlation, r = −0.336; p&amp;lt;0.01). Moreover, patients with high miR-223 expression demonstrated a significantly poorer prognosis than those with low expression (p=0.034). Based on a series of gain-of-function and loss-of-function investigation in vitro, we found that these effects may be due to the downregulation of the tumor-suppressor FBXW7, which was targeted by miR-223. Conclusions: Our present study indicates that high expression of miR-223 had a significant adverse impact on the survival of esophageal squamous cell carcinoma patients through repression of the function of FBXW7. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2306. doi:1538-7445.AM2012-2306

AMPK Activation by Dihydrotestosterone Reduces FSH-Stimulated Cell Proliferation in Rat Granulosa Cells by Inhibiting ERK Signaling Pathway

We have previously reported that 5α-dihydrotestosterone (DHT) inhibits FSH-mediated granulosa cell proliferation by reducing cyclin D2 mRNA expression and blocking cell cycle progression at G1/S phase. The present study investigated the role of AMP activated protein kinase (AMPK) in DHT-mediated inhibition of granulosa cell proliferation. Granulosa cells harvested from 3-d estradiol primed immature rats were exposed to different concentrations of DHT (0, 45, and 90 ng/ml) for 24 h. Western blot analysis of immunoprecipitated AMPK showed a dose-dependent activation (P < 0.05) as evidenced by the increased phosphorylation at thr 172. In addition, time-courses studies (0, 6, 12, and 24 h) using DHT (90 ng/ml) showed a time-dependent increase in AMPK activation with maximum effect at 24 h. FSH inhibited AMPK phosphorylation and promoted granulosa cell proliferation, but pretreatment with DHT (90 ng/ml) for 24 h prior to FSH treatment reduced this effect. Pharmacological activation of AMPK with 5-aminoimidazole-4-carboxamide-1-β4-ribofuranoside abolished FSH-mediated ERK phosphorylation, indicating that AMPK is a negative upstream regulator of ERK. Furthermore, inhibition of AMPK activation by compound C reversed the DHT-mediated reduction in positive cell cycle regulator, cyclin D2, and 5-bromo-2'-deoxyuridine incorporation. These results suggest that elevated levels of DHT activate AMPK, which in turn inhibits ERK phosphorylation. Thus, inhibition of ERK phosphorylation by activated AMPK in response to DHT might contribute to decreased granulosa cell mitogenesis and ovulatory dysfunction seen in hyperandrogenic states.

The CDC25B phosphatase shortens the G2 phase of neural progenitors and promotes efficient neuron production

During embryonic development, changes in cell cycle kinetics have been associated with neurogenesis. This observation suggests that specific cell cycle regulators may be recruited to modify cell cycle dynamics and influence the decision between proliferation and differentiation. In the present study, we investigate the role of core positive cell cycle regulators, the CDC25 phosphatases, in this process. We report that, in the developing chicken spinal cord, only CDC25A is expressed in domains where neural progenitors undergo proliferative self-renewing divisions, whereas the combinatorial expression of CDC25A and CDC25B correlates remarkably well with areas where neurogenesis occurs. We also establish that neural progenitors expressing both CDC25A and CDC25B have a shorter G2 phase than those expressing CDC25A alone. We examine the functional relevance of these correlations using an RNAi-based method that allows us to knock down CDC25B efficiently and specifically. Reducing CDC25B expression results in a specific lengthening of the G2 phase, whereas the S-phase length and the total cell cycle time are not significantly modified. This modification of cell cycle kinetics is associated with a reduction in neuron production that is due to the altered conversion of proliferating neural progenitor cells to post-mitotic neurons. Thus, expression of CDC25B in neural progenitors has two functions: to change cell cycle kinetics and in particular G2-phase length and also to promote neuron production, identifying new roles for this phosphatase during neurogenesis.