BrdUrd Labeling Research Articles

Up-regulated MiR-27-3p promotes the G1-S phase transition by targeting inhibitor of growth family member 5 in osteosarcoma

ObjectiveMicroRNAs (miRNAs) play an essential role in regulating malignant progression of tumour cells by inhibiting translation or stability of messenger RNA. However, the expression pattern and regulatory mechanism of miR-27-3p in osteosarcoma remains unclear. MethodsWe examined the expression of miR-27-3p in 5 osteosarcoma cell lines compared with that in 2 normal osteocyte cell lines. Osteosarcoma cells U-2OS and MG-63 were transduced to up-regulate or down-regulate the expression of miR-27-3p. The 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide, or MTT, assay, colony formation assays, BrdUrd labelling, immunofluorescence, anchorage-independent growth ability assay and flow cytometry analysis were used to test the effect of miR-27-3p. Luciferase assays were added to verify the direct relationship between miR-27-3p and the predicted target gene inhibitor of growth family member 5 (ING5). ResultsThe expression of miR-27-3p was significantly increased in examined osteosarcoma cell lines compared with that in normal osteocyte cell lines. Up-regulation of miR-27-3p significantly accelerated osteosarcoma cell growth via promoting G1-S transition. In addition, the opposite result was observed in miR-27-3p-down-regulated cells. Up-regulation of ING5 significantly attenuated the miR-27-3p-induced proliferation in osteosarcoma cells. ConclusionsThese data suggested that miR-27-3p could promote the G1-S phase transition that leads to proliferation by down-regulating the expression of ING5 in osteosarcoma.

TAZ inhibition restores sensitivity of cisplatin via AKT/mTOR signaling in lung adenocarcinoma.

The cisplatin-based doublet remains the foundation of treatment for the majority of patients with advanced non-small cell lung cancer (NSCLC), however, many patients exhibit acquired drug resistance. Transcriptional co-activator with PDZ binding motif (TAZ) is an oncogene in lung cancer, important for lung tumorigenesis and metastasis. Recently, it was revealed that TAZ upregulation confers resistance against a diverse range of cytotoxic agents. The present study aimed to investigate whether TAZ is involved in cisplatin sensitivity in lung adenocarcinoma. In the present study, we investigated TAZ expression in lung adenocarcinoma tissues and cell lines with western blot and RT-PCR analyses. By knocking down TAZ using short hairpin RNAs in cisplatin-resistant A549 and H460 cells, cell proliferation and apoptosis were assessed by BrdUrd labeling and flow cytometric analysis, respectively. The IC50 value of cisplatin was also assessed with CCK-8 assays. The western blotting for AKT, p-AKT, S6K and p-S6K were performed in cells with TAZ knockdown or overexpression. When the AKT/mTOR pathway was blocked in A549 and H460 with TAZ overexpression, cisplatin sensitivity was assessed by IC50 value. High expression of TAZ was found in lung adenocarcinoma tissues and cell lines, which were associated with cisplatin resistance. Knockdown of TAZ using shRNAs confered decreasing proliferation, increasing apoptosis and enhanced cisplatin sensitivity in cisplatin‑resistant cells. Additionally, TAZ knockdown decreased the AKT/mTOR pathway expression. Overexpression of TAZ increased p-AKT and p-S6K, which was inhibited by siAKT. Furthermore, we found that the inhibition of the AKT/mTOR pathway rescued the cells from cisplatin resistance caused by TAZ overexpression. Our data revealed that TAZ inhibition restores sensitivity of cisplatin in lung adenocarcinoma, which was, at least in part, AKT/mTOR signaling pathway-dependent. TAZ may be a potent therapeutic target for NSCLC in combination with conventional chemotherapy.

Abstract 2456: Mechanism of pancreatic cancer induction by smoking

Abstract Background & Aims: Cigarette smoking is a major risk factor for pancreatic cancer. However, the molecular signaling mechanisms of cigarette smoking remain poorly understood. We studied the effects of the cigarette smoking extract (CSE) and the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on apoptosis, proliferation and autophagy and underlying signaling mechanisms both in vitro in human pancreatic ductal cells and in vivo in mouse pancreas. We also studied the effect of K-ras mutation on the smoking-induced cell survival and proliferation. Methods: Immortalized human ductal cells HPDE6 were cultured for up to 1 week in the presence or absence of CSE (0.04-4µg/ml) or NNK (1nM-1µM). Pancreas-specific K-ras transgenic mice EL-K-ras were used as a cancer model. Wild type and EL-K-ras mice were injected with NNK (100mg/Kg) once weekly for 4 weeks and animals sacrificed 72h after the last injection. Apoptosis was assessed by measuring DNA fragmentation with Cell death ELISA or TUNEL and proliferation by 3H-thymidine incorporation into DNA or BrdUrd labeling. Autophagy was measured with its marker LC3 using immunofluorescence staining. Molecular signaling mechanisms were evaluated with Western. Results: Exposure of human ductal cells to CSE and NNK for 24h to 1 week time-dependently decreased apoptosis and autophagy and increased proliferation. Exposure to CSE and NNK decreased AMPK phosphorylation and increased Akt phosphorylation. Akt activation occurred at the times of AMPK inhibition. Pharmacological Akt inactivation reversed the inhibition of AMPK phosphorylation by CSE and NNK and prevented the effects of CSE and NNK on apoptosis and autophagy, indicating a central role of Akt in the prosurvival effects of tobacco compounds. In K-ras mice model of pancreatic cancer NNK injections induced increase in Akt kinase and decrease in AMPK phosphorylation. NNK decreased autophagy and apoptosis and stimulated proliferation in the pancreas. The NNK inhibitory effect on apoptosis and autophagy and the stimulation of proliferation were more pronounced in K-ras mice compared to wild type mice. Conclusion: Our in vitro and in vivo data indicate that smoking compounds create conditions favorable for cancer promotion through activating survival pathways including activation of Akt kinase and inhibition of AMPK activities; this leads to a decrease in apoptosis and autophagy and stimulation of proliferation. Our data suggest that smoking works in concert with K-ras mutation to induce adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2456.

The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice

JAK2 plays important roles in the regulation of a variety of cellular processes including cell migration, proliferation, and protection from apoptosis. Recently the L611S point mutation in JAK2 has been identified in a child with acute lymphoblastic leukemia. Here we analyzed the mechanism by which JAK2 exhibits its oncogenicity. In BaF3 murine hematopoietic cells, L611S mutant increased the expression of antiapoptotic proteins including X chromosome-linked inhibitor of apoptosis protein, inhibitor of apoptosis protein, and Bcl-XL. We also showed that JAK2 L611S mutant protects BaF3 cells from cytokine withdrawal-induced apoptotic cell death and leads to cytokine-independent cell growth. Furthermore BaF3 cells expressing JAK2 L611S mutant gained the ability to induce tumorigenesis in nude mice. The L611S mutant also exhibited malignancy, including prompt invasion and spreading into various organs, leading to rapid lethality of the mice. Finally we showed that a specific JAK2 inhibitor, AG490, potently inhibited cytokine-independent cell growth induced by JAK2 L611S mutant via the induction of apoptotic cell death. In addition, treatment with AG490 significantly inhibited the JAK2 L611S mutant-induced tumorigenesis in nude mice. Thus, our results both in vitro and in vivo strongly suggest that L611S mutant of JAK2 harbors potent oncogenic activity, and this probably requires the antiapoptotic signaling pathway.

Quantitative analysis of spermatogenic DNA synthesis in the rat using a monoclonal anti-5-bromodeoxyuridine antibody.

The utility of the 5-bromodeoxyuridine (BrdUrd) labelling technique for the quantitative analysis of spermatogenic deoxyribonucleic acid (DNA) synthesis was investigated in the rat. Rat testicles were labelled by a single intraperitoneal injection of 100 mg kg-1 of BrdUrd. The testicles were removed 1 h after injection, fixed in Bouin's fluid and embedded in paraffin. BrdUrd-labelled cells were detected by immunohistochemical staining using a monoclonal anti-BrdUrd antibody. The number of BrdUrd-labelled tubules per total number of tubules (percent L.T.), the number of BrdUrd-labelled cells per total number of tubules (tubular ratio) and the number of BrdUrd-labelled cells per number of Sertoli cells (Sertoli cell ratio in BrdUrd-labelled cells) were calculated as indices of spermatogenic DNA synthesis during each stage of the seminiferous epithelial wave. BrdUrd labelling was found exclusively in the nuclei of spermatogonia and in preleptotene spermatocytes in the seminiferous epithelium. The percent L.T. was generally greater than 50%, except in stages VI, VII and XIV, and the tubular as well as Sertoli cell ratios in BrdUrd-labelled cells was greater than 2.0 and 0.15, respectively, in stages I, II-III, V, VIII, X, and XII. The tubular ratio and Sertoli cell ratio in BrdUrd-labelled cells along the seminiferous epithelial wave had two distinct peaks. The distribution of the tubular ratio using the BrdUrd-labelling technique correlated well with the distribution previously established by measuring tritiated thymidine uptake per tubule. Thus, the BrdUrd labelling technique, which is more efficient than the tritiated thymidine labelling technique, can be used to quantitatively evaluate spermatogenic DNA synthesis.

Versican/PG-M Assembles Hyaluronan into Extracellular Matrix and Inhibits CD44-mediated Signaling toward Premature Senescence in Embryonic Fibroblasts

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix which interacts with hyaluronan at the N-terminal G1 domain, composed of A, B, and B' subdomains. Recently, we generated knock-in mice Cspg2(Delta3/Delta3), whose versican, without the A subdomain, has decreased hyaluronan (HA) binding affinity, thereby exhibiting reduced deposition of versican in the extracellular matrix. Here, we show that the Cspg2(Delta3/Delta3) fibroblasts within 20 passages proliferate more slowly and acquire senescence. Whereas the extracellular matrix of the wild type fibroblasts exhibited a network structure of hyaluronan and versican, that of the Cspg2(Delta3/Delta3) fibroblasts exhibited approximately 35 and approximately 85% deposition of versican and HA, without such a structure. The Cspg2(Delta3/Delta3) fibroblasts showed a substantial increase of ERK1/2 phosphorylation and expression of senescence markers p53, p21, and p16. Treatment of wild type fibroblasts with hyaluronidase and exogenous hyaluronan enhanced ERK1/2 phosphorylation, and treatment with an anti-CD44 antibody that blocks HA-CD44 interaction inhibited the phosphorylation. These results demonstrate that versican is essential for matrix assembly involving hyaluronan and that diminished versican deposition increases free hyaluronan fragments that interact with CD44 and increase phosphorylation of ERK1/2, leading to cellular senescence.

Up-regulating Sphingosine 1-Phosphate Receptor-2 Signaling Impairs Chemotactic, Wound-healing, and Morphogenetic Responses in Senescent Endothelial Cells

Vascular endothelial cells (ECs) have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest state called "cellular senescence." It has been shown that sphingolipids may be involved in senescence; however, the molecular links involved are poorly understood. In this study, we investigated the signaling and functions of sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid, in ECs of different in vitro ages. We observed that S1P-regulated responses are significantly inhibited and the S1P(1-3) receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P(1) and S1P(2) was also observed in the lesion regions of atherosclerotic endothelium, where senescent ECs have been identified in vivo. S1P-induced Akt and ERK1/2 activation were comparable between ECs of different in vitro ages; however, PTEN (phosphatase and tensin homolog deleted on chromosome 10) activity was significantly elevated and Rac activation was inhibited in senescent ECs. Rac activation and senescent-associated impairments were restored in senescent ECs by the expression of dominant-negative PTEN and by knocking down S1P(2) receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P(2) to a level similar to that of in vitro senescence. These results indicate that the impairment of function in senescent ECs in culture is mediated by an increase in S1P signaling through S1P(2)-mediated activation of the lipid phosphatase PTEN.

Leptin Increases Adult Hippocampal Neurogenesis in Vivo and in Vitro

Leptin, an adipose-derived hormone, has been implicated in several physiological processes involving the hippocampus. However, the role of leptin in adult hippocampal neurogenesis remains unknown. Here we show that leptin regulates neurogenesis in the dentate gyrus of adult mice as well as in cultured adult hippocampal progenitor cells. Chronic administration of leptin to adult mice increased cell proliferation without significant effects on the differentiation and the survival of newly proliferated cells in the dentate gyrus. The expression of the long form leptin receptor, LepRb, was detected in hippocampal progenitor cells by reverse transcription-PCR and immunohistochemistry. Leptin treatment also increased proliferation of cultured adult hippocampal progenitor cells. Analysis of signal transduction pathways revealed that leptin stimulated phosphorylation of Akt and STAT3 but not ERK1/2. Furthermore, pre-treating the cells with specific inhibitors of Akt or STAT3 attenuated leptin-induced cell proliferation in a dose-dependent manner. Taken together, our results support a role for leptin in adult hippocampal neurogenesis and suggest the involvement of the Akt and STAT3 signaling pathways in mediating the actions of leptin on neurogenesis.

Determinants That Control the Distinct Subcellular Localization of p38α-PRAK and p38β-PRAK Complexes

p38alpha and p38beta MAPKs (mitogen-activated protein kinases) share about 80% of their protein sequence identity, but have quite different biological functions. One such difference is in regulating the subcellular localization of their downstream kinases, such as PRAK (p38-regulated/activated protein kinase or MK5). The p38alpha-PRAK complex is found in the nucleus, whereas the p38beta-PRAK complex is exclusively localized to the cytosol. By generating a series of chimeric and point mutants of p38alpha and p38beta, we found two amino acid residues (Asp(145) and Leu(156) in p38alpha, Gly(145) and Val(156) in p38beta) that determine the distinct subcellular locations of p38alpha-PRAK and p38beta-PRAK. The subcellular localization of MK2 (MAPK-activated protein kinase 2), another downstream kinase of p38, was regulated in the same manner as that of PRAK. We found that nuclear import, but not export, determines the subcellular localization of p38alpha-PRAK and p38beta-PRAK. The published structure of the p38alpha-MK2 complex suggests Leu(156) of p38alpha is involved in the interaction with the nuclear localization signal in PRAK. The difference at this residue between p38alpha and p38beta may affect the nuclear localization signal in PRAK differently, and thereby influence the import of the complexes. Asp(145) in p38alpha (or Gly(145) in p38beta) is located on a different surface patch, and further random mutagenesis revealed that mutation of Asp(145), Thr(123), and Gln(325), the residues that can directly interact with importin alpha as predicted by modeling, but not mutation of the other 7 amino acid residues that cannot reach importin alpha, re-locate p38alpha-PRAK to the cytosol, suggesting that interaction with import machinery is involved in determining the subcellular localization of the p38alpha-PRAK and p38beta-PRAK complexes. Last, we show that nuclear localization of PRAK is required for its role in inhibiting the proliferation of NIH3T3 cells. In conclusion, multiple determinants control the distinct subcellular localization of p38alpha-PRAK and p38beta-PRAK complexes, and the location of PRAK plays a role in its function.

Transcription Factor Epiprofin Is Essential for Tooth Morphogenesis by Regulating Epithelial Cell Fate and Tooth Number

In tooth morphogenesis, the dental epithelium and mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth. We previously identified epiprofin (Epfn), a gene preferentially expressed in dental epithelia, differentiated ameloblasts, and certain ectodermal organs. To identify the role of Epfn in tooth development, we created Epfn-deficient mice (Epfn-/-). Epfn-/- mice developed an excess number of teeth, enamel deficiency, defects in cusp and root formation, and abnormal dentin structure. Mutant tooth germs formed multiple dental epithelial buds into the mesenchyme. In Epfn-/- molars, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Formation of the enamel knot, a signaling center for cusps, whose cells differentiate from the dental epithelium, was also inhibited. However, multiple premature nonproliferating enamel knot-like structures were formed ectopically. These dental epithelial abnormalities were accompanied by dysregulation of Lef-1, which is required for the normal transition from the bud to cap stage. Transfection of an Epfn vector promoted dental epithelial cell differentiation into ameloblasts and activated promoter activity of the enamel matrix ameloblastin gene. Our results suggest that in Epfn-deficient teeth, ectopic nonproliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.

Immobilization of Bioactive Fibroblast Growth Factor-2 into Cubic Proteinous Microcrystals (Bombyx mori Cypovirus Polyhedra) That Are Insoluble in a Physiological Cellular Environment

The supramolecular architecture of the extracellular matrix and the disposition of its specific accessory molecules give rise to variable heterotopic signaling cues for single cells. Here we have described the successful occlusion of human fibroblast growth factor-2 (FGF-2) into the cubic inclusion bodies (FGF-2 polyhedra) of the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). The polyhedra are proteinous cubic crystals of several microns in size that are insoluble in the extracellular milieu. Purified FGF-2 polyhedra were found to stimulate proliferation and phosphorylation of p44/p42 mitogen-activated protein kinase in cultured fibroblasts. Moreover, cellular responses were blocked by a synthetic inhibitor of the FGF signaling pathway, SU5402, suggesting that FGF-2 polyhedra indeed act through FGF receptors. Furthermore, FGF-2 polyhedra retained potent growth stimulatory properties even after desiccation. We have demonstrated that BmCPV polyhedra microcrystals that occlude extracellular signaling proteins are a novel and versatile tool that can be employed to analyze cellular behavior at the single cell level.

Critical Role for Transcription Coactivator Peroxisome Proliferator-activated Receptor (PPAR)-binding Protein/TRAP220 in Liver Regeneration and PPARα Ligand-induced Liver Tumor Development

Disruption of the gene encoding for the transcription coactivator peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/TRAP220/DRIP205/Med1) in the mouse results in embryonic lethality. Here, we have reported that targeted disruption of the Pbp/Pparbp gene in hepatocytes (Pbp(DeltaLiv)) impairs liver regeneration with low survival after partial hepatectomy. Analysis of cell cycle progression suggests a defective exit from quiescence, reduced BrdUrd incorporation, and diminished entry into G(2)/M phase in Pbp(DeltaLiv) hepatocytes after partial hepatectomy. Pbp(DeltaLiv) hepatocytes failed to respond to hepatocyte growth factor/scatter factor, implying that hepatic PBP deficiency affects c-met signaling. Pbp gene disruption also abolishes primary mitogen-induced liver cell proliferative response. Striking abrogation of CCl(4)-induced hepatocellular proliferation and hepatotoxicity occurred in Pbp(DeltaLiv) mice pretreated with phenobarbital due to lack of expression of xenobiotic metabolizing enzymes necessary for CCl(4) activation. Pbp(DeltaLiv) mice, chronically exposed to Wy-14,643, a PPARalpha ligand, revealed a striking proliferative response and clonal expansion of a few Pbp(fl/fl) hepatocytes that escaped Cre-mediated gene deletion in Pbp(DeltaLiv) livers, but no proliferative expansion of PBP null hepatocytes was observed. In these Pbp(DeltaLiv) mice, none of the Wy-14,643-induced hepatic adenomas and hepatocellular carcinomas was derived from PBP(DeltaLiv) hepatocytes; all liver tumors developing in Pbp(DeltaLiv) mice maintained non-recombinant Pbp alleles and retained PBP expression. These studies provide direct evidence in support of a critical role of PBP/TRAP220 in liver regeneration, induction of hepatotoxicity, and hepatocarcinogenesis.

SOX6 Suppresses Cyclin D1 Promoter Activity by Interacting with β-Catenin and Histone Deacetylase 1, and Its Down-regulation Induces Pancreatic β-Cell Proliferation

Sex-determining region Y-box (SOX) 6 negatively regulates glucose-stimulated insulin secretion from beta-cells and is a down-regulated transcription factor in the pancreatic islet cells of hyperinsulinemic obese mice. To determine the contribution of SOX6 to insulin resistance, we analyzed the effects of SOX6 on cell proliferation. Small interfering RNA-mediated attenuation of SOX6 expression stimulated the proliferation of insulinoma INS-1E and NIH-3T3 cells, whereas retroviral overexpression resulted in inhibition of cell growth. Quantitative real time-PCR analysis revealed that the levels of cyclin D1 transcripts were markedly decreased by SOX6 overexpression. Luciferase-reporter assay with beta-catenin showed that SOX6 suppresses cyclin D1 promoter activities. In vitro binding experiments showed that the LZ/Q domain of SOX6 physically interacts with armadillo repeats 1-4 of beta-catenin. Furthermore, chromatin immunoprecipitation assay revealed that increased SOX6 expression significantly reduced the levels of acetylated histones H3 and H4 at the cyclin D1 promoter. By using a histone deacetylase (HDAC) inhibitor and co-immunoprecipitation analysis, we showed that SOX6 suppressed cyclin D1 activities by interacting withbeta-catenin and HDAC1. The data presented suggest that SOX6 may be an important factor in obesity-related insulin resistance.

Inhibition of GSK3 Promotes Replication and Survival of Pancreatic Beta Cells

Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.

Trans-differentiation of Alveolar Epithelial Type II Cells to Type I Cells Involves Autocrine Signaling by Transforming Growth Factor β1 through the Smad Pathway

Trans-differentiation of Alveolar Epithelial Type II Cells to Type I Cells Involves Autocrine Signaling by Transforming Growth Factor β1 through the Smad Pathway

Rac1 GTPase Regulates Cell Genomic Stability and Senescence

The Rho family small GTPase Rac1 has been shown to play multiple roles in cell regulation, including actin cytoskeleton organization, transcriptional activation, microtubule dynamics, and endocytosis. Here, we report a novel role of Rac1 in regulating genomic stability and cell senescence. We observed in primary mouse embryonic fibroblasts that deletion of rac1 by gene targeting, as well as expression of the constitutively active Rac1 mutant L61Rac1, led to decreased cell growth that was associated with altered cell cycle progression at both G(1)/S and G(2)/M phases, increased apoptosis, and premature senescence. The senescence induction by either loss or gain of Rac1 activity was due at least in part to an increase in cellular reactive oxygen species (ROS). rac1 gene deletion caused a compensatory up-regulation of a closely related family member, Rac3, in mouse embryonic fibroblasts, the activity of which induced ROS production independently of Rac1. Furthermore, the Rac1-regulated ROS production and senescence correlated with the extent of DNA damage in the Rac1(-/-) and L61Rac1 cells. Treatment of these cells with a ROS inhibitor inhibited phospho-H2AX-positive nuclear focus formation. Finally, phospho-Ser(15) p53 was significantly increased in L61Rac1 and Rac1(-/-) cells, and genetic deletion of p53 from these cells readily reversed the senescence phenotype, indicating that Rac1 is functionally dependent on p53 in regulating cell senescence. Taken together, our results show that Rac1 activity serves as a regulator of cell senescence through modulation of cellular ROS, genomic stability, and p53 activity.

Granulocyte–Macrophage Colony-Stimulating Factor Is Essential for Normal Wound Healing

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent growth factor, which plays an important role during the process of wound healing. In clinical settings it has occasionally been employed in the treatment of cutaneous wounds of diverse etiologies. In a previous study, we have shown the positive influence of GM-CSF on full thickness excisional wounds in transgenic mice overexpressing GM-CSF in the basal layer of the epidermis. Direct GM-CSF action as well as indirect processes through the induction of secondary cytokines were proposed to contribute towards the beneficial effects. In this study, we analyzed the process of wound healing in transgenic mice overexpressing a GM-CSF antagonist in the epidermis. These mice not only exhibited a delayed scab rejection and reepithelialization but also neovascularization was reduced. The newly formed tissue was of poor quality as exhibited by the presence of extensive fibrosis. We suggest that the presence of GM-CSF in the repair process is of basic importance and its absence leads not only to delayed wound healing but it is also detrimental for the quality of the newly formed tissue.

Age and Bromodeoxyuridine Labelling Index as Prognostic Factors in High-grade Gliomas Treated with Surgery and Radiotherapy

Aims To determine the prognostic value of proliferative potential and DNA ploidy in 72 brain tumours (36 grade III and 36 grade IV astrocytomas) using bromodeoxyuridine (BrdUrd) incorporation and flow cytometry. Material and methods All 72 patients underwent excision, mostly incomplete of the tumour. After surgery, eight patients received conventionally fractionated radiotherapy, 11 patients received accelerated radiotherapy, and 53 patients received hypofractionated radiotherapy. Tumour samples taken during surgery from each patient were incubated in vitro for 1 h at 37°C with BrdUrd using the high pressure oxygen method. The percentage of BrdUrd-labelled cells (BrdUrd labelling index [BrdUrd LI]), and the total DNA content were evaluated. Results The tumours showed variability in the BrdUrd LI values, which ranged from 0.3 to 19.1%. No difference was observed in mean BrdUrd LI between grade III and grade IV sub-groups. A significantly higher percentage of DNA aneuploidy was observed in grade III gliomas (69.4%) than in grade IV gliomas (52.8%). Univariate analysis showed that younger patients (≤51 years) ( P = 0.021) with grade III gliomas ( P = 0.030) and low tumour proliferation rate (BrdUrd LI ≤ 2.7%, P = 0.028) had significantly higher 5-year survival rates. Tumour ploidy had no influence on patients' survival ( P = 0.591). However, Cox multi-variate analysis showed that only age over 51 years, and high tumour proliferation rate (BrdUrd LI > 2.7%), were significant unfavourable prognostic factors in patient survival. Conclusion In this study, independent prognostic factors for patients with high-grade gliomas treated with surgery and post-operative radiotherapy are age and tumour proliferation rate assessed according to the BrdUrd LI.

Dynamics of Hypoxia, Proliferation and Apoptosis after Irradiation in a Murine Tumor Model

Proliferation and hypoxia affect the efficacy of radiotherapy, but radiation by itself also affects the tumor microenvironment. The purpose of this study was to analyze temporal and spatial changes in hypoxia, proliferation and apoptosis after irradiation (20 Gy) in cells of a murine adenocarcinoma tumor line (C38). The hypoxia marker pimonidazole was injected 1 h before irradiation to label cells that were hypoxic at the time of irradiation. The second hypoxia marker, CCI-103F, and the proliferation marker BrdUrd were given at 4, 8 and 28 h after irradiation. Apoptosis was detected by means of activated caspase 3 staining. After immunohistochemical staining, the tumor sections were scanned and analyzed with a semiautomatic image analysis system. The hypoxic fraction decreased from 22% in unirradiated tumors to 8% at both 8 h and 28 h after treatment (P < 0.01). Radiation did not significantly affect the fraction of perfused vessels, which was 95% in unirradiated tumors and 90% after treatment. At 8 h after irradiation, minimum values for the BrdUrd labeling index (LI) and maximum levels of apoptosis were detected. At 28 h after treatment, the BrdUrd labeling and density of apoptotic cells had returned to pretreatment levels. At this time, the cell density had decreased to 55% of the initial value and a proportion of the cells that were hypoxic at the time of irradiation (pimonidazole-stained) were proliferating (BrdUrd-labeled). These data indicate an increase in tumor oxygenation after irradiation. In addition, a decreased tumor cell density without a significant change in tumor blood perfusion (Hoechst labeling) was observed. Therefore, it is likely that in this tumor model the decrease in tumor cell hypoxia was caused by reduced oxygen consumption.

Role of estrogen receptor β in colonic epithelium

Several papers report that the colon is one of the tissues regulated by estrogen receptor (ER)beta. To better understand the physiological role of ERbeta in colonic tissue, we have compared morphology, proliferation, and differentiation of colonic epithelium in ERbeta-/- mice and WT littermates. BrdUrd labeling revealed that the number of proliferating cells was higher in ERbeta-/- mice and that the migration of labeled cells toward the luminal surface was faster in ERbeta-/- mice than in WT littermates. Additionally, in the absence of ERbeta, there was a decrease in apoptosis, which was measured by immunohistochemical staining of cleaved caspase-3. The state of differentiation of the colonic epithelial cells was studied by using epithelial markers. In ERbeta-/- mice, there was a significant decrease in the expression of the differentiation marker cytokeratin (CK)20 and in the cellular adhesion molecules alpha-catenin (an adherens junction protein) and plectin (a hemidesmosomal protein). These changes were also evident by electron microscopy as abnormalities in tight junctions and in the number and shape of desmosomes in ERbeta-/- mice. These findings suggest a role for ERbeta in the organization and architectural maintenance of the colon. Furthermore, our results indicate that the rapidly proliferating cells of the colonic epithelium in ERbeta-/- mice are lost by increased shedding and not by increased apoptosis. In this way, hyperproliferative cells that lack ERbeta do not form hyperplastic lesions and do not accumulate in the superficial epithelium.