Lower Cell Proliferation Rate Research Articles

Arabidopsis NUCLEOSTEMIN-LIKE 1 (NSN1) regulates cell cycling potentially by cooperating with nucleosome assembly protein AtNAP1;1

BackgroundIn mammals, nucleostemin (NS), a nucleolar GTPase, is involved in stem cell proliferation, embryogenesis and ribosome biogenesis. Arabidopsis NUCLEOSTEMIN-LIKE 1 (NSN1) has previously been shown to be essential for plant growth and development. However, the role of NSN1 in cell proliferation is largely unknown.ResultsUsing nsn1, a loss-of-function mutant of Arabidopsis NSN1, we investigated the function of NSN1 in plant cell proliferation and cell cycle regulation. Morphologically, nsn1 exhibited developmental defects in both leaves and roots, producing severely reduced vegetative organs with a much smaller number of cells than those in the wild type. Dynamic analysis of leaf and root growth revealed a lower cell proliferation rate and slower cell division in nsn1. Consistently, the transcriptional levels of key cell cycle genes, including those regulating the transition of G1-S and G2-M, were reduced drastically in nsn1. The introduction of CYCLIN B1::GUS into nsn1 resulted in confined expression of GUS in both the leaf primordia and root meristem, indicating that cell proliferation was hampered by the mutation of NSN1. Upon subjection to treatment with bleomycin and methyl methanesulfonate (MMS), nsn1 plants exhibited hypersensitivity to the genotoxic agents. In the nucleus, NSN1 interacted with nucleosome assembly protein1 (AtNAP1;1), a highly conserved histone chaperone functioning in cell proliferation. Notably, the N-terminal conserved domains of Arabidopsis NSN1 were critical for the physical interaction.ConclusionsAs a conserved homolog of mammalian nucleostemin, Arabidopsis NSN1 plays pivotal roles in embryogenesis and ribosome biogenesis. In this study, NSN1 was found to function as a positive regulator in cell cycle progression. The interaction between NSN1 and histone chaperone AtNAP1;1, and the high resemblance in sensitivity to genotoxics between nsn1 and atnap1;1 imply the indispensability of the two nuclear proteins for cell cycle regulation. This work provides an insight into the delicate control of cell proliferation through the cooperation of a GTP-binding protein with a nucleosome assembly/disassembly protein in Arabidopsis.

Regulatory mechanism of microRNA-128 in osteosarcoma tumorigenesis and evolution through targeting SASH1.

Osteosarcoma, which commonly occurs in young individuals, is a type of malignant tumor of growing bones. MicroRNAs (miRNAs) have been found to be involved in various cancer-related processes. In the present study, it was reported that miRNA-128 (miR-128) was overexpressed in pathological tissues from patients with osteosarcoma. The present study investigated the possible regulatory mechanism of miR-128 on the progression of osteosarcoma and offered a foundation for clinical therapeutics in osteosarcoma. First, the expressions levels of miR-128 and its target gene, SAM and SH3 domain-containing 1 (SASH1), were measured in tissues from patients with osteosarcoma, and their correlation with osteosarcoma in terms of the pathological level were examined. The effects of miR-128 on osteosarcoma cell proliferation and apoptosis were examined, and its regulation of the expression levels of SASH1 and associated proteins was analyzed. Subsequently, the association between SASH1 and miR-128 was evaluated using a dual luciferase gene reporter assay. Finally, an in vivo xenograft tumor mouse model of osteosarcoma was established to confirm the in vitro results. The results demonstrated a higher expression of miR-128 in pathological tissues, compared with that in normal tissues. From examining the patient osteosarcoma tissues, marked correlations were found between the expression of miR-128 and that of SASH1, particularly with tumor size, invasion depth, lymph node metastasis, and tumor-node-metastasis stage. Compared with the negative control group and blank control group, the results showed that the inhibition of miR-128 led to a lower cell proliferation rate and higher apoptotic rate in MG-63 cells (P<0.05). Additionally, the expression of B-cell lymphoma 2 (Bcl-2) was downregulated in the miR-128-inhibited group, compared with that in the control group, whereas the expression levels of SASH1, Bcl-2-associated X protein and caspase-3 were upregulated in the group with miR-128 inhibition (P<0.05). SASH1 was confirmed as a direct target of miR-128 using a dual luciferase gene reporter assay. Finally, the downregulation of miR-128 was found to induce tumor suppressive effects on xenograft tumor models of osteosarcoma in mice in vivo. The results of the present study suggested that miR-128 may regulate the tumorigenesis and evolution of osteosarcoma through targeting SASH1.

Seasonal variation of cell proliferation and apoptosis in the efferent ductules and epididymis of the Neotropical bat Artibeus lituratus (Chiroptera, Phyllostomidae)

The balance between cell proliferation and apoptosis is important for maintenance of male fertility, being influenced by a variety of stimuli including androgens and estrogens. However, studies concerning regulation of these processes along the male reproductive tract under physiological conditions are scarce. Therefore, in this study, we investigated the profile of cell proliferation and apoptosis in the efferent ductules and epididymis of the Neotropical bat Artibeus lituratus, a seasonal breeder that presents natural variation in components of the androgen and estrogen responsive systems along the circannual cycle. Low rates of cell proliferation and apoptosis were found in the efferent ductules and epididymis of A. lituratus during the reproductive period, as few epithelial cells were positive for MCM7 (proliferation marker) and cleaved caspase-3 or TUNEL (apoptosis markers). In contrast, during the regressive period, the rate of both proliferating and apoptotic cells was significantly higher in the epithelium lining the efferent ductules as well as throughout the epididymis. The increased proliferative activity at this phase was positively correlated with the expression of estrogen receptor alpha (ERα), whereas the variation in apoptosis appears to be unrelated to the local expression of androgen and estrogen receptors. Together, these data suggest that cell proliferation and apoptosis are differentially modulated in the efferent ductules and epididymis of A. lituratus during the annual reproductive cycle, and support the hypothesis that ERα may be important in preparing the male reproductive tract for sexual recrudescence.

Comparisons between silk fibroin nonwoven electrospun fabrics using aqueous and formic acid solutions

ABSTRACTThe objective of this study is to compare the spinnability, morphology, structure, mechanical properties, and cell compatibility of the silk fibroin nanofiber nonwoven electrospun fabrics using aqueous (AQ) solution and formic acid (FA) solution. The lower limit concentration was 5 wt% and 3 wt% of AQ solution and FA solution for electrospinning, respectively. The fiber diameter of electrospun fabric using FA solution was larger than that using aqueous solution at the same concentration. The secondary structure contents of silk fabrics were same between AQ and FA solutions. FA was remained in silk nanofibers, and the remained FA could be neutralized. Young’s modulus and cell adhesion on electrospun fabric using FA was lower than that using AQ solution. On the contrary, lower cell proliferation rate on electrospun fabric using FA was kept even after neutralization.

Cascading effect in bioprocessing-The impact of mild hypothermia on CHO cell behavior and host cell protein composition.

A major challenge in downstream purification of monoclonal antibodies (mAb) is the removal of host cell proteins (HCPs). Previous studies have shown that cell culture conditions significantly impact the HCP content at harvest. However, it is currently unclear how process conditions affect physiological changes in the host cell population, and how these changes, in turn, cascade down to change the HCP profile. We examined how temperature downshift (TDS) to mild hypothermia affects key upstream performance indicators, that is antibody titre, HCP concentration and HCP species, across the cell culture decline phase and at harvest through the lens of changes in cellular behavior. Mild hypothermic conditions introduced on day 5 of fed-batch Chinese hamster ovary (CHO) cell bioreactors resulted in a lower cell proliferation rate but larger percentages of healthier cells across the cell culture decline phase compared to bioreactors maintained at standard physiological temperature. Moreover, the onset of apoptosis was less evident in mild hypothermic cultures. Consequently, mild hypothermic cultures took an extra 5 days to reach an integral viable cell concentration (IVCC) and antibody yield similar to that of the control at standard physiological temperature. When cell viability dropped below 80%, mild hypothermic cell cultures had a reduced variety of HCP species by 36%, including approximately 44% and 27% lower proteases and chaperones, respectively, despite having similar HCP concentration. This study suggests that TDS may be a good strategy to provide cleaner downstream feedstocks by reducing the variety of HCPs and to maintain product integrity by reducing the number of proteases and chaperones.

STIP Regulates ERK1/2 Signaling Pathway Involved in Interaction with PP1γ in Lymphoblastic Leukemia.

Sip1/Tuftelin Interacting Protein (STIP) is highly conserved from Caenorhabditis elegans to Homo sapiens and has essential biological functions. However, its function in leukemia remains unknown. Clinic samples and cell model were used in this article to investigate the expression of STIP in lymphoblastic leukemia. The functional research of STIP was performed in ARH-77 by siRNA transfection, immunofluorescence, cell count, cell cycle analysis, qRT-PCR, sub-cellular fractionation assays, immunoprecipitation and western blotting. Here, we found that STIP is more highly expressed in both clinical lymphoblastic leukemia samples and cultured leukemia cells than in normal samples. Knockdown of STIP in B lymphoblastic leukemia ARH-77 cells leads to S phase arrest, lower cell proliferation rates, and suppressed AKT and ERK1/2 signaling pathways. Interestingly, when protein phosphatase was inhibited by Calyculin A, STIP knockdown did not result in the dephosphorylation of p-ERK1/2, suggesting the dependence of STIP on protein phosphatase in the regulation of ERK1/2. Among those protein phosphatase inhibited by Calyculin A, PP1γ was found to interact with STIP proven by immunofluorescence and immunoprecipitation assays. The binding of STIP with PP1γ may decrease the phosphatase activity of PP1γ, resulting in hyper-activated ERK1/2 signaling. In summary, the high expression and activation effect on the ERK1/2 signaling of STIP in lymphoblastic leukemia suggest that STIP would be a potential therapy target or diagnosis marker for leukemia.

Radiofrequency hyperthermia promotes the therapeutic effects on chemotherapeutic-resistant breast cancer when combined with heat shock protein promoter-controlled HSV-TK gene therapy: Toward imaging-guided interventional gene therapy.

ObjectiveGene therapy is a frontier in modern medicine. In the present study, we explored a new technique for the effective treatment of multidrug-resistant (MDR) breast cancer by combining fully the advantages of multidisciplinary fields, including image-guided minimally invasive interventional oncology, radiofrequency technology, and direct intratumoral gene therapy.ResultsCombination treatment with PHSP-TK plus RFH resulted in significantly higher TK gene transfection/expression, as well as a lower cell proliferation rate and a higher cell apoptosis index, than those of control groups. In vivo validation experiments with MRI confirmed that combination therapy resulted in a significant reduction of relative tumor volume compared with those of control animals, which was supported by the results of histologic and apoptosis analyses.Materials and methodsThe heat shock protein promoter (PHSP) was used to precisely control the overexpression of thymidine kinase (TK) (PHSP-TK). Serial in vitro experiments were performed to confirm whether radiofrequency hyperthermia (RFH) could enhance PHSP-TK transfection and expression in a MDR breast cancer cell line (MCF7/Adr). Serial in vivo experiments were then carried out to validate the feasibility of the new technique, termed interventional RFH-enhanced direct intratumoral PHSP-TK gene therapy. The therapeutic effect of combination therapy was evaluated by MRI and confirmed by subsequent laboratory correlation.ConclusionsThis study has established “proof-of-principle” of a new technique, interventional RFH-enhanced local gene therapy for MDR breast cancer, which may open new avenues for the effective management of MDR breast cancers via the simultaneous integration of interventional oncology, RF technology, and direct intratumoral gene therapy.

MiR-26b enhances radiosensitivity of hepatocellular carcinoma cells by targeting EphA2.

Although low-dose radiotherapy (RT) that involves low collateral damage is more suitable for hepatocellular carcinoma (HCC) than traditional high-dose RT, but to achieve satisfactory therapeutic effect with low-dose RT, it is necessary to sensitize HCC cells to irradiation. This study was aimed to determine whether radiosensitivity of HCC cells can be enhanced using miR-26b by targeting erythropoietin producing human hepatocelluar A2 (EphA2). The levels of miR-26b and EphA2 expression in multiple HCC cell lines were assessed by qPCR and western blotting, respectively, and compared with those in a hepatic cell line. HCC 97H cells were transfected with miR-26b mimics, EphA2-ShRNA or EphA2 over-expression vector before exposure to low-dose irradiation. Different degrees of miR-26b down-regulation and EphA2 up-regulation were observed in all HCC cell lines, among which the HCC 97H cell line expressed the lowest level of miR-26b and highest level of EphA2. EphA2 was verified as the target of miR-26b by dual luciferase reporter assay. HCC 97H cells transfected with miR-26b mimics or EphA2-ShRNA reduced the expression of EphA2 protein, with significantly lower cell proliferation rate and cell invasion ability and higher apoptosis rate in response to low-dose irradiation than those in the non-transfected cells. These results were reversed after EphA2 was overexpressed by transfection with the EphA2 overexpression vector. Co-transfection with miR-26b mimics and EphA2 overexpression vector barely altered EphA2 expression level and cell response to low-dose irradiation. These data suggest that miR-26b enhances radiosensitivity of HCC 97H cells by targeting EphA2 protein.

Hepatic B cell leukemia-3 suppresses chemically-induced hepatocarcinogenesis in mice through altered MAPK and NF-κB activation.

The transcriptional nuclear factor kappa B (NF-κB)-coactivator B cell leukemia-3 (Bcl-3) is a molecular regulator of cell death and proliferation. Bcl-3 has been shown to be widely expressed in different cancer types including hepatocellular carcinoma (HCC). Its influence on hepatocarcinogenesis is still undetermined. To examine the role of Bcl-3 in hepatocarcinogenesis mice with hepatocyte-specific overexpression of Bcl-3 (Bcl-3Hep) were exposed to diethylnitrosamine (DEN) and phenobarbital (PB). Hepatic Bcl-3 overexpression attenuated DEN/PB-induced hepatocarcinogenesis. Bcl-3Hep mice exhibited a lower number and smaller tumor nodules in response to DEN/PB at 40 weeks of age. Reduced HCC formation was accompanied by a lower rate of cell proliferation and a distinct expression pattern of growth and differentiation-related genes. Activation of c-Jun N-terminal kinase (JNK) and especially extracellular-signal regulated kinase (ERK) was reduced in tumor and tumor-surrounding liver tissue of Bcl-3Hep mice, while p38 and NF-κB p65 were phosphorylated to a higher extent compared to the wild type. In parallel, the absolute number of intrahepatic macrophages, CD8+ T cells and activated B cells was reduced in DEN/PB-treated Bcl-3Hep mice mirroring a reduction of tumor-associated inflammation. Interestingly, at the early time point of 7 weeks following tumor initiation, a higher rate of apoptotic cell death was observed in Bcl-3Hep mice. In summary, hepatocyte-restricted Bcl-3 overexpression reduced hepatocarcinogenesis related to prolonged liver injury early after tumor initiation likely due to decreased survival of DEN/PB-damaged, premalignant cells. Therefore, Bcl-3 could become a novel player in the development of therapeutic and diagnostic tools for HCC.

Cerium oxide nanoparticles stimulate proliferation of primary mouse embryonic fibroblasts in vitro

The increasing application of cell therapy technologies in the treatment of various diseases requires the development of new effective methods for culturing primary cells. The major limitation for the efficient use of autologous cell material is the low rate of cell proliferation. Successful cell therapy requires sufficient amounts of cell material over a short period of time with the preservation of their differentiation and proliferative potential. In this regard, the development of novel, highly efficient stimulators of proliferative activity in stem cells is a truly urgent task. In this paper we have demonstrated that citrate-stabilized cerium oxide nanoparticles (nanoceria) enhance the proliferative activity of primary mouse embryonic fibroblasts in vitro. Cerium oxide nanoparticles stimulate cell proliferation in a wide range of concentrations (10−3М–10−9M) through reduction of intracellular levels of reactive oxygen species (ROS) during the lag phase of cell growth and by modulating the expression level of the major antioxidant enzymes. We found the optimal concentration of nanoceria, which provides the greatest acceleration of cell proliferation in vitro, while maintaining the levels of intracellular ROS and mRNA of antioxidant enzymes in the physiological range. Our results confirm that nanocrystalline ceria can be considered as a basis for effective and inexpensive supplements in cell culturing.

VprBP, an HIV target protein, is vital for immune responses by controlling T cell growth, proliferation and senescence.

Abstract Naïve mature T cells are maintained in a quiescent state with a low rate of cell size growth (biomass increase due to enhanced metabolism and biosynthesis) and proliferation. Disturbances inflicted by infection and inflammation, often trigger cells to exit quiescence and enter cell cycle, followed by rapid proliferation to restore homeostasis. Cell size growth is imperative for proliferating cells to compensate for the halving of cellular constituents for each division. Thus, proper coordination of cell growth and cell cycle entry of T cells is essential to mount effective immune response. Nonetheless, little is known about how cell cycle entry is controlled and even less about how cell size growth is regulated in T cells. We studied protein expression during T cell growth and cell cycle entry using quantitative proteomic approach. VprBP, an HIV target protein, was identified to be one of the highly upregulated factors. We found that VprBP was required for the cell growth and cell cycle entry of activated T cells. VprBP deletion abrogated T cell expansion during antiviral and autoimmune responses in vivo. VprBP-deficient cells drastically stabilized p53 protein with elevated expression of p53 target genes. Intriguingly, we found that although p53 deletion failed to correct the T cell growth defect due to VprBP deletion, it largely restored the cell cycle entry of VprBP-deficient T cells. In addition, VprBP deletion led to a premature senescence phenotype in T cells associating with increased cellular aging markers. These results suggest that VprBP controls T cell growth, cell cycle entry and senescence via discrete molecular programs. The recent findings and the underlying mechanisms will be discussed in detail in this presentation.

Identification of Pathways Mediating Growth Differentiation Factor5-Induced Tenogenic Differentiation in Human Bone Marrow Stromal Cells.

To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.

In vivo mechanisms of uterine myoma volume reduction with ulipristal acetate treatment

To study the in vivo mechanisms of action of ulipristal acetate (UPA) on uterine myomas. Retrospective histologic and immunohistochemical (IHC) study of myomas. Academic research unit. Among 59 women with symptomatic myomas who underwent myomectomy, 42 were treated preoperatively with UPA, while 17 were not. Histology and IHC were analyzed on tissue microarrays obtained from surgical specimens. Proliferation, apoptosis, extracellular matrix (ECM) remodeling, and matrix metalloproteinase 2 (MMP-2) expression. Proliferation was low in all conditions, with no statistical difference between groups. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay showed an increase in cell death in UPA-treated myomas compared with untreated myomas, but only after short-term treatment; this was not associated with elevated levels of cleaved caspase-3. After long-term treatment, cell density was higher and the ECM volume fraction lower in UPA-treated myomas than in untreated myomas. MMP-2 expression was found to be increased after treatment, showing the highest level after long-term treatment, compared with untreated myomas. Regarding sustained clinical volume reduction of myomas, this study strongly points to multifactorial mechanisms of action of UPA, involving: 1) a persistently low cell proliferation rate; 2) a limited period of cell death; and 3) ECM remodeling concomitant with stimulation of MMP-2 expression.

Transcriptome sequencing reveals differences between anagen and telogen secondary hair follicle-derived dermal papilla cells of the Cashmere goat (Capra hircus)

Dermal papilla is considered the control center of hair follicle growth and hair cycle. The secondary hair follicle (producing cashmere) growth cycle of the Cashmere goat (Capra hircus) is circannual, and each growth phase can be easily distinguished by its long duration. To identify gene expression patterns and differences of the dermal papilla cell (DPC) between the anagen and telogen phases, we established two DPC lines: ana-DPCs (DPCs derived from the anagen secondary hair follicle) and tel-DPCs (DPCs derived from the telogen secondary hair follicle). Compared with the ana-DPCs, the tel-DPCs lost the capacity to form cell aggregates and showed lower cell proliferation rate. Transcriptome sequencing revealed that 825 genes were differentially expressed by at least threefold between the two DPC lines. These genes were significantly enriched in cell cycle control, cell division, and chromosome partitioning from the Eukaryotic Orthologous Groups of proteins (KOG) database and in cell cycle, cell adhesion molecules, cytokine-cytokine receptor interaction, and p53 signaling pathway from the Kyoto Encyclopedia of Gene and Genomes (KEGG) database. Enrichment analyses revealed that in the middle of the telogen the DPCs of secondary hair follicles (SHFs) seemed on the one hand to promote the degeneration of SHFs and cessation of cashmere growth, while on the other hand to resist self-apoptosis and prepare for the regeneration or revivification of fully functional dermal papillae. These findings provide a better understanding of hair follicle growth and will be useful for identification of novel molecules associated with the control of hair growth cycle.

Isolation, characterization and the multi‐lineage differentiation potential of rabbit bone marrow‐derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are recognized by their plastic adherent ability, fibroblastic-like appearance, expression of specific surface protein markers, and are defined by their ability to undergo multi-lineage differentiation. Although rabbit bone marrow-derived MSCs (rbMSCs) have been used extensively in previous studies especially in translational research, these cells have neither been defined morphologically and ultrastructurally, nor been compared with their counterparts in humans in their multi-lineage differentiation ability. A study was therefore conducted to define the morphology, surface marker proteins, ultrastructure and multi-lineage differentiation ability of rbMSCs. Herein, the primary rbMSC cultures of three adult New Zealand white rabbits (at least 4 months old) were used for three independent experiments. rbMSCs were isolated using the gradient-centrifugation method, an established technique for human MSCs (hMSCs) isolation. Cells were characterized by phase contrast microscopy observation, transmission electron microscopy analysis, reverse transcriptase-polymerase chain reaction (PCR) analysis, immunocytochemistry staining, flow cytometry, alamarBlue(®) assay, histological staining and quantitative (q)PCR analysis. The isolated plastic adherent cells were in fibroblastic spindle-shape and possessed eccentric, irregular-shaped nuclei as well as rich inner cytoplasmic zones similar to that of hMSCs. The rbMSCs expressed CD29, CD44, CD73, CD81, CD90 and CD166, but were negative (or dim positive) for CD34, CD45, CD117 and HLD-DR. Despite having similar morphology and phenotypic expression, rbMSCs possessed significantly larger cell size but had a lower proliferation rate as compared with hMSCs. Using established protocols to differentiate hMSCs, rbMSCs underwent osteogenic, adipogenic and chondrogenic differentiation. Interestingly, differentiated rbMSCs demonstrated higher levels of osteogenic (Runx2) and chondrogenic (Sox9) gene expressions than that of hMSCs (P < 0.05). There was, however, no difference in the adipogenic (Pparγ) expressions between these cell types (P > 0.05). rbMSCs possess similar morphological characteristics to hMSCs, but have a higher potential for osteogenic and chondrogenic differentiation, despite having a lower cell proliferation rate than hMSCs. The characteristics reported here may be used as a comprehensive set of criteria to define or characterize rbMSCs.

Nm23-H1 gene driven by hTERT promoter induces inhibition of invasive phenotype and metastasis of lung cancer xenograft in mice.

Lung cancer is the leading cause of cancer death in both men and women worldwide. Tumor metastasis is an essential aspect of lung cancer progression and patient death. The nm23-H1 gene has been extensively investigated as a metastasis suppressor gene. Our previous studies have revealed: that a significant relationship exists between the low-level expression nm23-H1 in primary non-small cell lung cancer (NSCLC) with increased metastasis and a poor prognosis; that L9981-nm23-H1 cells (a nm23-H1 transfactant cell) exhibited lower cell proliferation rates, more G0/G1 phase growth, and an increase in apoptosis with a dramatic decrease in the tumor cells' ability to invade than L9981 cells did; and that L9981- nm23-H1 cells also demonstrated a significantly reduced lymph node and distant metastatic capacity in vivo than L9981 cells did in nude mice. In this study, we construct a plasmid containing the nm23-H1 gene, which was driven by the human telomerase reverse transcriptase (hTERT) promoter. We evaluated the anti-invasion and anti-metastatic effects of pGL3-hTP-nm23 on L9981, a human large cell lung cancer cell line with nm23-H1 negative expression, by transwell assay in vitro and bioluminescence in nude mice models. The toxicity of pGL3-hTP-nm23 and its effects on tumor growth were evaluated in nude mice models after gene therapy. The cell cycles, apoptosis, and proliferation of the nm23-H1 transfactant were also detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay) and flow cytometry (FCM). The results showed that the hTERT-promoter dramatically drives nm23-H1 gene expression, and induces inhibition of cell growth and migration in L9981-luc cells and MRC-5 cells in vitro. nm23-H1 also significantly inhibited the tumorigenesis and distant metastasis of L9981-luc cell in vivo. Moreover, no obvious side effect was detected in normal mouse tissues after intratumoral injection of the vector. The treatment of the nm23-H1 gene driven by hTERT promoter appears to be a promising approach for the gene therapy of nm23-H1 low-expressed tumors.

Expressão de MMPs, marcadores angiogênicos e proliferação celular em tumores odontogênicos

INTRODUÇÃO E OBJETIVO: O conhecimento do comportamento biológico de lesões de natureza odontogênica é essencial para tornar a abordagem terapêutica adequada e estabelecer um prognóstico. A produção de metaloproteinases da matriz extracelular (MMPs), a angiogênese e a proliferação celular fornecem subsídios para o crescimento tumoral. O presente artigo tem como objetivo fazer uma revisão de literatura de pesquisas em tumores odontogênicos (TOs) selecionados a partir da nova classificação da Organização Mundial da Saúde (OMS) de 2005 sobre a expressão de MMPs, marcadores angiogênicos e proliferação celular e verificar, nestes estudos, a relação desses marcadores quanto ao comportamento biológico dessas lesões. RESULTADOS: Nota-se que as MMPs -1, -2, -7, -9 e -26 encontram-se mais expressas no componente epitelial e estroma e, particularmente, a -13 em estroma. Uma maior angiogênese é observada em TOs mais agressivos. CD 105 foi mais expresso no TO ceratocístico (TOC) e CD34 em ameloblastomas sólidos (ASs). Relata-se elevada expressão do Ki-67 e p53 no TOC e no AS e baixo índice de proliferação celular no TO adenomatoide (TOA). CONCLUSÃO: Esses resultados mostram que as MMPs participam no processo de invasão e recorrência de algumas lesões odontogênicas, estando associadas ao comportamento biológico desses tumores. A angiogênese é fundamental para fornecer suporte à proliferação celular e esses dois eventos em conjunto estão correlacionados com diferentes níveis de comportamento biológico nos TOs, quando comparados com cistos de natureza odontogênica, o que pode sugerir o uso de inibidores angiogênicos como provável abordagem terapêutica nessas lesões.

757 HYPERTENSION-RELATED, CALCIUM-REGULATED GENE (HCaRG/COMMD5) INHIBITS KIDNEY CANCER DEVELOPMENT

Introduction: Hypertension is an individual risk factor not only for renal failure but also for renal cell carcinoma (RCC). Hypertension-related, calcium-regulated gene (HCaRG/COMMD5)is more expressed in kidneys of the genetically hypertensive rats than normotensive controls. Transgenic mice overexpressing HCaRG in proximal tubules are more resistant to ischemic kidney injury. HCaRG accelerates tubular repair after injury by controlling cell proliferation and facilitating re-differentiation through the induction of p21 via p53-independent pathway. HCaRG expression is lower in RCCs compared to normal adjacent tissues in humans. Hypothesis: HCaRG could be a factor linking hypertension with the risk of RCC. Methods: To elucidate whether HCaRG can inhibit RCC development, we examined the effect of HCaRG overexpression in Renca cells (murine RCC cell line) and a murine RCC homograft model. Results: HCaRG-overexpressing Renca cells exhibited lower cell proliferation rate and more differentiated phenotype compared to Neo-Renca cells. Experimental RCC derived from HCaRG-Renca cells were significantly smaller than tumors of Neo-Renca cells after 10 days of subcutaneous implantation. In the HCaRG-overexpressing tumors, PI3K/Akt and MAPK pathways were inhibited compared to controls. HCaRG overexpression dramatically decreased the expression of EGFR and ErbB3, thus inhibiting the phosphorylation of HER2. Tumor angiogenesis was also reduced in HCaRG-overexpressing tumors and serum VEGF concentrations were lower in mice implanted with HCaRG-Renca cells. Conclusion: HCaRG overexpression inactivated ErbB pathway by inhibiting the expression of EGFR and HER3, and the phosphorylation of HER2, resulting in reduced RCC progression. HCaRG deficiencies may be involved in the risk of RCC in hypertensive patients.

Endoplasmic reticulum factor ERLIN2 regulates cytosolic lipid content in cancer cells

Increased de novo lipogenesis is a hallmark of aggressive cancers. Lipid droplets, the major form of cytosolic lipid storage, have been implicated in cancer cell proliferation and tumorigenesis. Recently, we identified the ERLIN2 [ER (endoplasmic reticulum) lipid raft-associated 2) gene that is amplified and overexpressed in aggressive human breast cancer. Previous studies demonstrated that ERLIN2 plays a supporting oncogenic role by facilitating the transformation of human breast cancer cells. In the present study, we found that ERLIN2 supports cancer cell growth by regulating cytosolic lipid droplet production. ERLIN2 is preferably expressed in human breast cancer cells or hepatoma cells and is inducible by insulin signalling or when cells are cultured in lipoprotein-deficient medium. Increased expression of ERLIN2 promotes the accumulation of cytosolic lipid droplets in breast cancer cells or hepatoma cells in response to insulin or overload of unsaturated fatty acids. ERLIN2 regulates activation of SREBP (sterol regulatory element-binding protein) 1c, the key regulator of de novo lipogenesis, in cancer cells. ERLIN2 was found to bind to INSIG1 (insulin-induced gene 1), a key ER membrane protein that blocks SREBP activation. Consistent with the role of ERLIN2in regulating cytosolic lipid content, down-regulation of ERLIN2in breast cancer or hepatoma cells led to lower cell proliferation rates. The present study revealed a novel role for ERLIN2in supporting cancer cell growth by promoting the activation of the key lipogenic regulator SREBP1c and the production of cytosolic lipid droplets. The identification of ERLIN2 as a regulator of cytosolic lipid content in cancer cells has important implications for understanding the molecular basis of tumorigenesis and the treatment of cancer.

The influence of pore size on osteoblast phenotype expression in cultures grown on porous titanium

This study investigated the effect of pore size on osteoblastic phenotype development in cultures grown on porous titanium (Ti). Porous Ti discs with three different pore sizes, 312μm (Ti 312), 130μm (Ti 130) and 62μm (Ti 62) were fabricated using a powder metallurgy process. Osteoblastic cells obtained from human alveolar bone were cultured on porous Ti samples for periods of up to 14 days. Cell proliferation was affected by pore size at day 3 (p=0.0010), day 7 (p=0.0005) and day 10 (p=0.0090) in the following way: Ti 62<Ti 130<Ti 312. Gene expression of bone markers evaluated at 14 days was affected, RUNX2 (p=0.0153), ALP (p=0.0153), BSP (p=0.0156), COL (p=0.0156), and OPN (p=0.0156) by pore size as follows: Ti 312<Ti 130<Ti 62. Based on these results, the authors suggest that porous Ti surfaces with pore sizes near 62μm, compared with those of 312μm and 130μm, yield the highest expression of osteoblast phenotype as indicated by the lower cell proliferation rate and higher gene expression of bone markers.