Cell Proliferation In Association Research Articles

GREB1 isoform 4 is specifically transcribed by MITF and required for melanoma proliferation

Growth regulation by estrogen in breast cancer 1 (GREB1) is involved in hormone-dependent and -independent tumor development (e.g., hepatoblastoma). In this study, we found that a GREB1 splicing variant, isoform 4 (Is4), which encodes C-terminal half of full-length GREB1, is specifically expressed via microphthalmia-associated transcription factor (MITF) in melanocytic melanoma, and that two MITF-binding E-box CANNTG motifs at the 5’-upstream region of GREB1 exon 19 are necessary for GREB1 Is4 transcription. MITF and GREB1 Is4 were strongly co-expressed in approximately 20% of the melanoma specimens evaluated (17/89 cases) and their expression was associated with tumor thickness. GREB1 Is4 silencing reduced melanoma cell proliferation in association with altered expression of cell proliferation-related genes in vitro. In addition, GREB1 Is4 targeting by antisense oligonucleotide (ASO) decreased melanoma xenograft tumor formation and GREB1 Is4 expression in a BRAFV600E; PTENflox melanoma mouse model promoted melanoma formation, demonstrating the crucial role of GREB1 Is4 for melanoma proliferation in vivo. GREB1 Is4 bound to CAD, the rate-limiting enzyme of pyrimidine metabolism, and metabolic flux analysis revealed that GREBI Is4 is necessary for pyrimidine synthesis. These results suggest that MITF-dependent GREB1 Is4 expression leads to melanoma proliferation and GREB1 Is4 represents a new molecular target in melanoma.

Extracellular vesicles from focal segmental glomerulosclerosis pediatric patients induce STAT3 activation and mesangial cell proliferation.

Primary focal segmental glomerulosclerosis (FSGS), a major cause of end-stage kidney disease (ESKD) in adolescents and young adults, is attributable to recognized genetic mutations in a minority of cases. For the majority with idiopathic primary FSGS, the cause of the disease is unknown. We hypothesize that extracellular vesicle (EVs), that carry information between podocytes and mesangial cells, may play a key role in disease progression. A total of 30 participants (20 primary nephrotic syndrome/ 10 healthy controls) were enrolled in this study. Primary nephrotic syndrome subjects were grouped based on pathologic diagnosis. The FSGS group was compared to healthy control subjects based on demographic and clinical findings. EVs were isolated from the urine of each group before being characterized by Western blotting, transmission electron microscopy, and nanoparticle tracking analysis. The effects of the EVs from each group on normal human mesangial cells and activation of certain pathways were then investigated. Based on demographic and clinical findings, mean serum creatinine was significantly higher in the FSGS group than the normal healthy control group. The mean size of the EVs in the FSGS group was significantly higher than the healthy control group. The mesangial cells that were challenged with EVs isolated from FSGS patients showed significant upregulation of STAT-3, PCNA, Ki67, and cell proliferation. Our data demonstrate that EVs from FSGS patients stimulate mesangial cell proliferation in association with upregulation of the phospho-STAT-3 pathway. Additional studies are planned to identify the molecular cargo within the EVs from FSGS patients that contribute to the pathogenesis of FSGS.

Glyphosate-based herbicide enhances the uterine sensitivity to estradiol in rats.

In a previous work, we detected that postnatal exposure to a glyphosate-based herbicide (GBH) alters uterine development in prepubertal rats causing endometrial hyperplasia and increasing cell proliferation. Our goal was to determine whether exposure to low-dose of a GBH during postnatal development might enhance the sensitivity of the uterus to an estrogenic treatment. Female Wistar pups were subcutaneously injected with saline solution (control) or GBH using the reference dose (2 mg/kg/day, EPA) on postnatal days (PND) 1, 3, 5, and 7. At weaning (PND21), female rats were bilaterally ovariectomized and treated with silastic capsules containing 17β-estradiol (E2, 1mg/ml) until they were two months of age. On PND60, uterine samples were removed and processed for histology, immunohistochemistry and mRNA extraction to evaluate: i) uterine morphology, ii) uterine cell proliferation by the detection of Ki67, iii) the expression of the estrogen receptors alpha (ESR1) and beta (ESR2), and iv) the expression of WNT7A and β-catenin. GBH-exposed animals showed increased luminal epithelial height and stromal nuclei density. The luminal and glandular epithelium were markedly hyperplastic in 43% of GBH-exposed animals. GBH exposure caused an increase in E2-induced cell proliferation in association with an induction of both ESR1 and ESR2. GBH treatment decreased membranous and cytoplasmic expression of β-catenin in luminal and glandular epithelial cells and increased WNT7A expression in the luminal epithelium. These results suggest that early postnatal exposure to a GBH enhances the sensitivity of the rat uterus to estradiol, and induces histomorphological and molecular changes associated with uterine hyperplasia.

Yes-associated protein mediates human embryonic stem cell-derived cardiomyocyte proliferation: Involvement of epidermal growth factor receptor signaling.

Unlike mature cardiomyocytes, human pluripotent stem cell-derived cardiomyocytes exhibit higher proliferative capacity; however, the underlying mechanisms involved are yet to be elucidated. Here, we revealed that the Yes-associated protein (YAP) plays a critical role in regulating cell proliferation in association with epidermal growth factor receptor (EGFR) in human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Our results show that low-density culture significantly promotes the proliferation of hESC-CMs via YAP. Interestingly, the low-density culture-induced YAP expression further induced EGFR expression, without any alterations in the activity of EGFR and its two major downstream kinases, ERK, and AKT. However, treatment of a low-density-culture of hESC-CMs with epidermal growth factor (EGF) increased proliferation via phosphorylation of EGFR, ERK, and AKT, and the EGF-induced phosphorylation of EGFR, ERK, and AKT was significantly higher in low-density hESC-CMs than in high-density hESC-CMs. Furthermore, the EGF-induced activation of EGFR, ERK, and AKT increased YAP expression and subsequently proliferation. In conclusion, YAP mediates both low-density culture-induced and EGF-induced proliferation of hESC-CMs in low-density culture conditions.

Segmentation and tracking of stem cells in time lapse microscopy to quantify dynamic behavioral changes during spheroid formation.

Dynamic behavior of stem cells during in vitro development is diverse. Previous cell tracking studies have focused more on cell proliferation than on cell aggregation. However, the enhancement of cell proliferation in association with cell aggregation has been reported. In a previous study, we also demonstrated that the aggregation of adult human mesenchymal stem cells to form three-dimensional (3D) cellular spheroids helped maintain the expression of stemness marker genes in the cells. However, the dynamic behavioral changes triggered by spheroid formation remain to be investigated. A scheme of image processing techniques is proposed to meet this need. A hybrid-thresholding technique was first developed for efficient segmentation of cell clusters, after which a cell tracking method based on pair-matching with topological constraints was designed. Two morphological indices were derived to track the timing of 3D spheroid formation during the cellular aggregation process. Five cell motility indices measured from single cells and 3D spheroids were then compared. After confirmation of more than 90% correspondence between the results obtained by manual tracking and the proposed methods, an analysis of cellular behavior reveals a significant increase in motility in association with spheroid formation, consistent with a previous report that used a gene expression approach. This study proposed a systematic image analysis method to quantify the dynamic behavior of stem cells for stemness evaluation during cell culturing in vitro. Results demonstrated the validity of the developed platform in investigation of the dynamic behavior of cell aggregation in stem cell cultures in vitro.

Depletion of C3orf1/TIMMDC1 inhibits migration and proliferation in 95D lung carcinoma cells.

In our previous study, we identified an association of high expression of c3orf1, also known as TIMMDC1 (translocase of inner mitochondrial membrane domain-containing protein 1), with metastatic characteristics in lung carcinoma cells. To investigate the preliminary function and mechanism of this mitochondrial protein, we depleted C3orf1 expression by introducing siRNA into 95D lung carcinoma cells. We demonstrated that C3orf1 depletion significantly suppressed 95D cell growth and migration. We confirmed C3orf1 localization in the inner mitochondrial membrane and showed that mitochondrial viability, membrane potential, and ATPase activity were remarkably reduced upon depletion of C3orf1. Microarray data indicated that genes involved in regulation of cell death, migration, and cell-cycle arrest were significantly altered after C3orf1 depletion for 48 h. The expression of genes involved in focal adhesion, ECM-receptor interaction, and p53-signaling pathways were notably altered. Furthermore, cell-cycle arrest genes such as CCNG2 and PTEN as well as genes involved in cell migration inhibition, such as TIMP3 and COL3A1, were upregulated after C3orf1 depletion in 95D cells. Concurrently, expression of the migration-promoting gene NUPR1 was markedly reduced, as confirmed by real-time PCR. We conclude that C3orf1 is critical for mitochondrial function, migration, and proliferation in 95D lung carcinoma cells. Depletion of C3orf1 inhibited cell migration and cell proliferation in association with upregulation of genes involved in cell-cycle arrest and cell migration inhibition. These results suggest that C3orf1 (TIMMDC1) may be a viable treatment target for lung carcinoma, and that further study of the role of this protein in lung carcinoma pathogenesis is justified.

Abstract 3713: Chemoprevention of prostate carcinogenesis in TRAMP mice by α-santalol by causing cell cycle arrest and induction of apoptosis.

Abstract α-santalol, a major component of sandalwood oil inhibits growth of cultured prostate cancer cells in vitro by causing apoptosis. The present study was undertaken to determine the in vivo efficacy of α-santalol using TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice as a model. The i.p administration of α-santalol (50 mg/kg) did not cause any side effects or weight loss throughout the study. Administration of α-santalol (50 mg/kg) showed a trend in decrease in average wet weights of urogenital weights compared to control mice (∼34% decrease in α-santalol group compared to control). Furthermore, the dorsolateral sections of prostate from α-santalol-treated mice exhibited decreased cell proliferation in association with induction of apoptosis. Western blotting analysis of prostate tumor samples from α-santalol-treated group revealed an increase in the expression levels of cleaved PARP, phospho p53 (ser 15), cleaved caspase-3, cyclin B, p21 and a modest reduction in antiapoptotic protein Bcl-xL compared to control samples. In agreement with these results, α-santalol treatment resulted in G2/M cell cycle arrest in cultured prostate cancer cells. In conclusion, the present study indicates that α-santalol administration inhibits the development of prostate cancer in TRAMP mice by decreasing cell proliferation, causing cell cycle arrest, and inducing apoptosis. This study was supported by Wilkes University Type I grant, American Association of Colleges of Pharmacy and by Translational Cancer Research Center funded by South Dakota Governor's Office of Economic Development. Citation Format: Ajay Bommareddy, William Eggelston, Stacy Prelewicz, Andrea Antal, Adam L. VanWert, Hiral Patel, Aleona Chinikaylo, Linda S. Gutierrez, Santha Sreevidya, Chandradhar Dwivedi. Chemoprevention of prostate carcinogenesis in TRAMP mice by α-santalol by causing cell cycle arrest and induction of apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3713. doi:10.1158/1538-7445.AM2013-3713

Anti-Myeloma Effects of the Janus Kinase 2 (JAK2) Inhibitor SAR503 Alone and in Combination Treatment

AbstractAbstract 4986 Background:Janus kinase 2 (JAK2) is a cytoplasmic tyrosine kinase that carries out a series of cascading signals via signal transducer and activator of transcription (STAT)s, mitogen-activated protein kinase (MAPK), and phosphorylation of PI3K. Activation of the JAK2 pathway plays an important role in both normal and malignant hematopoiesis. The JAK pathway ha been shown to play a key role in multiple myeloma (MM). JAK2 has been specifically implicated in signaling by members of the type II cytokine receptor family (interferon [IFN] receptor), GM-CSF receptor (IL-3R, IL-5R, and GM-CSF-R), gp130 receptor family interleukin-6 (IL-6R) and single chain receptors (Epo-R, Tpo-R, GH-R, and PRL-R). IFN-α inhibits MM cell proliferation in association with cell cycle arrest at G1 and limits the clonogenic growth of both MM cell lines and primary MM patient specimens. SAR503 (Sanofi-Aventis) is a potent, highly selective JAK2 inhibitor. Thus, we evaluated the anti-MM effects of SAR503 as a single agent and in combination with other anti-MM drugs and evaluated gene and protein expression in MM cells exposed to these drugs. Experiment design:The MM cell lines RPMI8226, U266, and MM1s were cultured in RPMI1640 with standard nutrition supplements. Bone marrow aspirates were obtained from MM patients following informed consent. Bone marrow mononuclear cells (BMMCs) were isolated by using density-gradient centrifugation with Histopaque-1077 (Sigma, St Louis). Cells were plated in 96 well plates at a concentration of 6 × 104 cells/100 ml/well, and incubated for 24 hours prior to drug treatment, after which time the drugs were added in replicates of six for 48 hours. BMMCs were incubated in the presence of media, SAR503, doxorubicin, melphalan, dexamethasone, bortezomib, or IFN-α alone or the combination of SAR503 with one of these anti-MM agents. Following the 48-hour drug incubation, cell viability was assessed utilizing the cell proliferation MTS assay. For gene expression studies, total RNA was isolated MM tumor cells with or without drug exposure. RNA was reverse-transcribed into cDNA and amplified using the Thermo-Script RT-PCR System and PCR performed again using the GeneAmp PCR System 9700. Protein phosphorylation of MM tumor cells with or without drug exposure was determined with Western blot analysis. Results:SAR503 alone inhibited MM tumor cell proliferation in a concentration-dependent fashion. The 50% growth inhibition (IC50) of cells from MM cell lines at 48 hours varied (IC50: RPMI8226 1mM; U266 0. 5mM; MM1s 10mM). IC50 of primary MM tumor cells treated with SAR503 ranged from approximately 5 to 10mM in different patients. Notably, the combination of SAR503 and either doxorubicin or melphalan showed markedly reduced cell viability compared to either drug alone in all three MM cell lines and primary tumor cells from MM patients. Since this effect may have resulted from decreased cell proliferation due to inhibition of the JAK2 pathway and cell cycle arrest or increased cell death, we further determined cell apoptosis of MM tumor cells treated with SAR503 alone by using flow cytometric analysis to detect Annexin V and propidium iodide (PI) staining. Our data showed SAR503 increased MM tumor cell apoptosis in a concentration-dependent fashion. The combination of SAR503 and dexamethasone or bortezomib only slightly reduced tumor cell viability in both MM cell lines and primary MM tumor cells more than single agent treatment, and the combination of SAR503 with IFN-α did not enhance the anti-MM effects compared to single drug treatment. Notably, RT-PCR results showed marked decreases in both AKT1 and mTOR gene expression in MM tumor cells treated with SAR503. Conclusion:The combination of the JAK2 inhibitor SAR503 with doxorubicin or melphalan markedly reduces MM tumor cell viability more than single agent treatment. The results from these studies suggest that enhanced anti-MM activity may be observed when SAR503 is combined with conventional treatment for MM. We are currently evaluating the anti-MM effects of SAR503 in these combination treatments in vivo using our MM xenograft models. Disclosures:Berenson:Onyx: Consultancy, Honoraria, Speakers Bureau.

Effects of voluntary running on plasma levels of neurotrophins, hippocampal cell proliferation and learning and memory in stressed rats

Previous studies have shown that a 2-week treatment with 40mg/kg corticosterone (CORT) in rats suppresses hippocampal neurogenesis and decreases hippocampal brain-derived neurotrophic factor (BDNF) levels and impairs spatial learning, all of which could be counteracted by voluntary wheel running. BDNF and insulin-like growth factor (IGF-1) have been suggested to mediate physical exercise-enhanced hippocampal neurogenesis and cognition. Here we examined whether such running-elicited benefits were accompanied by corresponding changes of peripheral BDNF and IGF-1 levels in a rat model of stress. We examined the effects of acute (5days) and chronic (4weeks) treatment with CORT and/or wheel running on (1) hippocampal cell proliferation, (2) spatial learning and memory and (3) plasma levels of BDNF and IGF-1. Acute CORT treatment improved spatial learning without altered cell proliferation compared to vehicle treatment. Acute CORT-treated non-runners showed an increased trend in plasma BDNF levels together with a significant increase in hippocampal BDNF levels. Acute running showed no effect on cognition, cell proliferation and peripheral BDNF and IGF-1 levels. Conversely, chronic CORT treatment in non-runners significantly impaired spatial learning and suppressed cell proliferation in association with a decreased trend in plasma BDNF level and a significant increase in hippocampal BDNF levels. Running counteracted cognitive deficit and restored hippocampal cell proliferation following chronic CORT treatment; but without corresponding changes in plasma BDNF and IGF-1 levels. The results suggest that the beneficial effects of acute stress on cognitive improvement may be mediated by BDNF-enhanced synaptic plasticity that is hippocampal cell proliferation-independent, whereas chronic stress may impair cognition by decreasing hippocampal cell proliferation and BDNF levels. Furthermore, the results indicate a trend in changes of plasma BDNF levels associated with a significant alteration in hippocampal levels, suggesting that treatment with running/CORT for 4 weeks may induce a change in central levels of hippocampal BDNF level, which may not lead to a significant change in peripheral levels.

Vaccarin promotes endothelial cell proliferation in association with neovascularization in vitro and in vivo.

Angiogenesis is a major pathological component of several diseases, including traumatic vascular disease and coronary heart disease. The purpose of the present study was to determine the effects of vaccarin on endothelial cell migration and neovascularization, which are important and necessary components of wound healing. The present study investigated and confirmed neovascularization induced by vaccarin in vitro and in vivo. In vitro, the effects of vaccarin (1.08 and 2.15 µM) on proliferation, migration and tube formation of human microvascular endothelial cells (HMEC)-1 were evaluated via sulforhodamine B assay and migration and tube formation assay, respectively. Furthermore, a mouse Matrigel plus model was used to detect capillary-like tube structures in vivo. Immunohistochemistry was used to detect the protein expression of cluster of differentiation 31 (CD31), p-AKT and p-extracellular-signal-regulated kinases (Erk). Vaccarin significantly promoted HMEC-1 proliferation and migration and tube formation of HMEC-1 at a dose of 2.15 µM. In vivo, vaccarin delivered by daily oral administration significantly improved epidermal growth factor-induced angiogenesis in an intradermal inoculation mouse model. The mouse Matrigel model study also revealed that vaccarin significantly promoted neovascularization via detection of CD31 levels and enhanced protein expression of p-Akt and p‑Erk. In addition, vaccarin also promoted expression of CD31.

Gene profile of myeloid-derived suppressive cells from the bone marrow of lysosomal acid lipase knock-out mice.

BackgroundLysosomal acid lipase (LAL) controls development and homeostasis of myeloid lineage cells. Loss of the lysosomal acid lipase (LAL) function leads to expansion of myeloid-derived suppressive cells (MDSCs) that cause myeloproliferative neoplasm.Methodology/Principal FindingsAffymetrix GeneChip microarray analysis identified detailed intrinsic defects in Ly6G+ myeloid lineage cells of LAL knock-out (lal−/−) mice. Ingenuity Pathway Analysis revealed activation of the mammalian target of rapamycin (mTOR) signaling, which functions as a nutrient/energy/redox sensor, and controls cell growth, cell cycle entry, cell survival, and cell motility. Loss of the LAL function led to major alteration of large GTPase and small GTPase signal transduction pathways. lal−/− Ly6G+ myeloid cells in the bone marrow showed substantial increase of cell proliferation in association with up-regulation of cyclin and cyclin-dependent kinase (cdk) genes. The epigenetic microenvironment was significantly changed due to the increased expression of multiple histone cluster genes, centromere protein genes and chromosome modification genes. Gene expression of bioenergetic pathways, including glycolysis, aerobic glycolysis, mitochondrial oxidative phosphorylation, and respiratory chain proteins, was also increased, while the mitochondrial function was impaired in lal−/− Ly6G+ myeloid cells. The concentration of reactive oxygen species (ROS) was significantly increased accompanied by up-regulation of nitric oxide/ROS production genes in these cells.Conclusions/SignificanceThis comprehensive gene profile study for the first time identifies and defines important gene pathways involved in the myeloid lineage cells towards MDSCs using lal−/− mouse model.

Fbxw7-dependent Degradation of Notch Is Required for Control of “Stemness” and Neuronal-Glial Differentiation in Neural Stem Cells

Control of the growth and differentiation of neural stem cells is fundamental to brain development and is largely dependent on the Notch signaling pathway. The mechanism by which the activity of Notch is regulated during brain development has remained unclear, however. Fbxw7 (also known as Fbw7, SEL-10, hCdc4, or hAgo) is the F-box protein subunit of an Skp1-Cul1-F-box protein (SCF)-type ubiquitin ligase complex that plays a central role in the degradation of Notch family members. We now show that mice with brain-specific deletion of Fbxw7 (Nestin-Cre/Fbxw7(F/F) mice) die shortly after birth with morphological abnormalities of the brain and the absence of suckling behavior. The maintenance of neural stem cells was sustained in association with the accumulation of Notch1 and Notch3, as well as up-regulation of Notch target genes in the mutant mice. Astrogenesis was also enhanced in the mutant mice in vivo, and the differentiation of neural progenitor cells was skewed toward astrocytes rather than neurons in vitro, with the latter effect being reversed by treatment of the cells with a pharmacological inhibitor of the Notch signaling pathway. Our results thus implicate Fbxw7 as a key regulator of the maintenance and differentiation of neural stem cells in the brain.

Inhibitory effect of fenofibrate on neointima hyperplasia via G 0/G 1 arrest of cell proliferation

We have previously reported that fenofibrate displayed a potent antithrombotic effect by the inhibition of platelet aggregation. The present study was designed to investigate the effects of fenofibrate on the neointimal hyperplasia and its possible molecular mechanism. Neointimal hyperplasia was measured in balloon-inflated–induced vascular injury model of male Sprague–Dawley rats and cell proliferation was measured in primary cultured rat aortic vascular smooth muscle cells (VSMCs). Fenofibrate-treated group showed a significant reduction in neointimal formation (0.07 ± 0.04 mm 2) from the control (0.13 ± 0.04 mm 2). Fenofibrate significantly inhibited platelet-derived growth factor (PDGF)-BB-induced cell counting and [ 3H]-thymidine incorporation into DNA. Fenofibrate suppressed the PDGF-BB-inducible progression through G 0/G 1 to S phase of cell cycle. Moreover, fenofibrate inhibited not only phosphorylation of retinoblastoma (Rb) protein and expression of cyclin D/E, CDK 2/4 and proliferating cell nuclear antigen (PCNA) proteins but also mitogen-activated protein kinase (MAPK) signaling pathways such as ERK 1/2, p38 and JNK phosphorylation. In conclusion, the present study demonstrates that fenofibrate significantly inhibits neointimal formation via G 0/G 1 arrest of PDGF-BB-induced cell proliferation in association with the inhibition of MAPK, which resulted in the downregulation of expressions of cyclin D/E, CDK 2/4 and PCNA proteins, suggesting that fenofibrate may be useful for individuals with a high risk of thrombotic or cardiovascular diseases.

Neurofibromatosis 2 with peripheral neuropathies: Electrophysiological, pathological and genetic studies of a Taiwanese family

The objective of this study was to assess peripheral nerve involvement and DNA mutation of the neurofibromatosis type 2 (NF2) gene (NF2) in a Taiwanese family with classic NF2. Eleven members (six symptomatic and five asymptomatic) of a family carrying NF2 underwent clinical examination, neuroimaging, and electrophysiological analysis. Mutation and linkage analyses were conducted on DNA samples prepared from peripheral blood (all individuals), a sural nerve biopsy specimen (one symptomatic member), and a tumor specimen (another symptomatic member). Six of the 11 members were diagnosed with classic NF2. DNA sequencing of the tumor specimen demonstrated a frameshift mutation with 756delC on exon 8 of NF2. Three affected subjects showed clinical variability of the neuropathic disorders. Electrophysiological studies demonstrated variation in the disease pattern and severity of peripheral nerve involvement in five affected subjects. The morphometric assessment of the sural nerve biopsy specimen showed a marked reduction in both large myelinated and unmyelinated fibre density and increased density of non-myelinating Schwann cell nuclei. Apart from numerous pathological nuclei of isolated Schwann cells, multiple profiles of non-myelinating Schwann cell subunits were apparent in the endoneurium. Schwann cell proliferation in association with first-hit mutation of the merlin gene might be responsible for the NF2-associated neuropathy. Sural nerve biopsy showed a progressive neuropathy in the disease. Further, we suggest nonmyelinating Schwann cells are involved in NF2 neuropathy.

Nicotine induces cell proliferation in association with cyclin D1 up-regulation and inhibits cell differentiation in association with p53 regulation in a murine pre-osteoblastic cell line

Recent studies have suggested that nicotine critically affects bone metabolism. Many studies have examined the effects of nicotine on proliferation and differentiation, but the underlying molecular mechanisms remain unclear. We examined cell cycle regulators involved in the proliferation and differentiation of MC3T3-E1 cells. Nicotine induced cell proliferation in association with p53 down-regulation and cyclin D1 up-regulation. In differentiated cells, nicotine reduced alkaline phosphatase activity and mineralized nodule formation in dose-dependent manners. Furthermore, p53 expression was sustained in nicotine-treated cells during differentiation. These findings indicate that nicotine promotes the cell cycle and inhibits differentiation in association with p53 regulation in pre-osteoblastic cells.

Zinc Supplementation Enhances Hepatic Regeneration by Preserving Hepatocyte Nuclear Factor-4α in Mice Subjected to Long-Term Ethanol Administration

Alcoholic liver disease is associated with sustained liver damage and impaired regeneration, as well as significant zinc deficiency. This study was undertaken to examine whether dietary zinc supplementation could improve liver regeneration by increasing the expression of genes involved in hepatic cellular proliferation in a mouse model of alcoholic liver disease. Adult 129S6 mice fed an ethanol-containing liquid diet for 6 months developed alcoholic liver disease as measured by serum alanine transferase activity and histopathological changes. Zinc supplementation to ethanol-exposed mice enhanced liver regeneration as indicated by increased numbers of proliferation cell nuclear antigen (PCNA)-positive and bromodeoxyuridine (BrdU)-labeled hepatocytes. Zinc-enhanced liver regeneration was associated with an increase in hepatocyte nuclear factor-4alpha (HNF-4alpha), a liver-enriched, zinc-finger transcription factor. Studies using cultured HepG2 cells showed that zinc deficiency suppressed cell proliferation and cell proliferation-related proteins, including hepatocyte growth factor (HGF), insulin-like growth factor I (IGF-I), insulin-like growth factor binding protein 1 (IGFBP1), metallothionein (MT), and cyclin D1, as well as HNF-4alpha. HNF-4alpha gene silencing inhibited cell proliferation in association with decreased protein levels of IGF-I, IGFBP1, MT, and cyclin D1. The present study provides evidence that zinc supplementation enhances liver regeneration at least in part by HNF-4alpha through the up-regulation of cell proliferation-related proteins, suggesting that dietary zinc supplementation may have beneficial effects in alcoholic liver disease.

The combination of novel low molecular weight inhibitors of RAF (LBT613) and target of rapamycin (RAD001) decreases glioma proliferation and invasion

Monotherapies have proven largely ineffective for the treatment of glioblastomas, suggesting that increased patient benefit may be achieved by combining therapies. Two protumorigenic pathways known to be active in glioblastoma include RAS/RAF/mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT/target of rapamycin (TOR). We investigated the efficacy of a combination of novel low molecular weight inhibitors LBT613 and RAD001 (everolimus), which were designed to target RAF and TOR, respectively. LBT613 decreased phosphorylation of extracellular signal-regulated kinase 1 and 2, downstream effectors of RAF, in a human glioma cell line. RAD001 resulted in decreased phosphorylation of the TOR effector S6. To determine if targeting RAF and TOR activities could result in decreased protumorigenic glioma cellular behaviors, we evaluated the abilities of LBT613 and RAD001 to affect the proliferation, migration, and invasion of human glioma cells. Treatment with either LBT613 or RAD001 alone significantly decreased the proliferation of multiple human glioma cell lines. Furthermore, LBT613 and RAD001 in combination synergized to decrease glioma cell proliferation in association with G(1) cell cycle arrest. Glioma invasion is a critical contributor to tumor malignancy. The combination of LBT613 and RAD001 inhibited the invasion of human glioma cells through Matrigel to a greater degree than treatment with either drug alone. These data suggest that the combination of LBT613 and RAD001 reduces glioma cell proliferation and invasion and support examination of the combination of RAF and TOR inhibitors for the treatment of human glioblastoma patients.

Endothelial cell–restricted disruption of FoxM1 impairs endothelial repair following LPS-induced vascular injury

Recovery of endothelial integrity after vascular injury is vital for endothelial barrier function and vascular homeostasis. However, little is known about the molecular mechanisms of endothelial barrier repair following injury. To investigate the functional role of forkhead box M1 (FoxM1) in the mechanism of endothelial repair, we generated endothelial cell-restricted FoxM1-deficient mice (FoxM1 CKO mice). These mutant mice were viable and exhibited no overt phenotype. However, in response to the inflammatory mediator LPS, FoxM1 CKO mice displayed significantly protracted increase in lung vascular permeability and markedly increased mortality. Following LPS-induced vascular injury, FoxM1 CKO lungs demonstrated impaired cell proliferation in association with sustained expression of p27(Kip1) and decreased expression of cyclin B1 and Cdc25C. Endothelial cells isolated from FoxM1 CKO lungs failed to proliferate, and siRNA-mediated suppression of FoxM1 expression in human endothelial cells resulted in defective cell cycle progression. Deletion of FoxM1 in endothelial cells induced decreased expression of cyclins, Cdc2, and Cdc25C, increased p27(Kip1) expression, and decreased Cdk activities. Thus, FoxM1 plays a critical role in the mechanism of the restoration of endothelial barrier function following vascular injury. These data suggest that impairment in FoxM1 activation may be an important determinant of the persistent vascular barrier leakiness and edema formation associated with inflammatory diseases.

The primary antimitotic mechanism of action of the synthetic halichondrin E7389 is suppression of microtubule growth

E7389, which is in phase I and II clinical trials, is a synthetic macrocyclic ketone analogue of the marine sponge natural product halichondrin B. Whereas its mechanism of action has not been fully elucidated, its main target seems to be tubulin and/or the microtubules responsible for the construction and proper function of the mitotic spindle. Like most microtubule-targeted antitumor drugs, it inhibits tumor cell proliferation in association with G(2)-M arrest. It binds to tubulin and inhibits microtubule polymerization. We examined the mechanism of action of E7389 with purified microtubules and in living cells and found that, unlike antimitotic drugs including vinblastine and paclitaxel that suppress both the shortening and growth phases of microtubule dynamic instability, E7389 seems to work by an end-poisoning mechanism that results predominantly in inhibition of microtubule growth, but not shortening, in association with sequestration of tubulin into aggregates. In living MCF7 cells at the concentration that half-maximally blocked cell proliferation and mitosis (1 nmol/L), E7389 did not affect the shortening events of microtubule dynamic instability nor the catastrophe or rescue frequencies, but it significantly suppressed the rate and extent of microtubule growth. Vinblastine, but not E7389, inhibited the dilution-induced microtubule disassembly rate. The results suggest that, at its lowest effective concentrations, E7389 may suppress mitosis by directly binding to microtubule ends as unliganded E7389 or by competition of E7389-induced tubulin aggregates with unliganded soluble tubulin for addition to growing microtubule ends. The result is formation of abnormal mitotic spindles that cannot pass the metaphase/anaphase checkpoint.

Activation of STAT3/Smad1 Is a Key Signaling Pathway for Progression to Glomerulosclerosis in Experimental Glomerulonephritis

Mesangial cell proliferation is a significant event in the development of progressive glomerular injuries. However, the issue of how cell proliferation is involved in the development of glomerulosclerosis is unclear. Recently, we showed that the overexpression of type IV collagen (Col IV), a major component of mesangial extracellular matrix, is transcriptionally regulated by Smad1 in diabetic glomerulosclerosis. In this study, we have demonstrated the effect of the administration of an anti-platelet-derived growth factor (PDGF) beta-receptor antibody (APB5) blocking activation by the PDGF-B chain on rat glomerulonephritis and have examined the signaling pathways that regulate both glomerular cell proliferation and glomerulosclerosis in vivo and in vitro. Experimental mesangial proliferative glomerulonephritis (Thy1 GN) was induced by a single intravenous injection of anti-rat Thy-1.1 monoclonal antibody. In Thy1 GN, mesangial cell proliferation and the expression of Col IV peaked at day 6. Immunohistochemical staining for the expression of Smad1, phospho-Smad1 (pSmad1), and phospho-STAT3 (pSTAT3) revealed that the peak for glomerular Smad1 expression occurred at day 6, consistent with the peak for mesangial proliferation. The expression of pSmad1 was up-regulated at day 1, and the peak for glomerular pSmad1 expression occurred at day 4 of the disease. When treated with APB5, both mesangial proliferation and sclerosis were reduced significantly. The expression of Smad1, pSmad1, and pSTAT3 was also significantly reduced by the administration of APB5. PDGF induced both mesangial cell replication and Col IV synthesis in association with an increased expression of pSTAT3 and pSmad1 on cultured mesangial cells. In addition, APB5 reduced mesangial cell proliferation in association with decreased pSmad1, pSTAT3, and Col IV protein expressions in vitro. The introduction of dominant negative STAT3 significantly decreased the expression of Col IV in cultured mesangial cells. These data suggest that the activation of STAT3 and Smad1 participates in the developing process of glomerulosclerosis in experimental glomerulonephritis.