Growth Related Molecules Research Articles

Polarization of human gingival fibroblasts by Th1-, Th2-, Th17-, and Treg-derived cytokines.

The purpose of this study was to evaluate whether gingival fibroblasts (GFs) can be differently activated and polarized into distinct functional subtypes by T-helper (Th) cytokines. Gingival fibroblasts were stimulated with interferon (IFN)-γ, interleukin (IL)-4, IL-17, and transforming growth factor (TGF)-β, representative cytokines of Th1, Th2, Th17, and regulatory T cells, respectively, and the gene expression profiles were analyzed by microarray. Differentially expressed genes (DEGs) in GFs stimulated by 4 cytokines were screened, and a gene ontology (GO) analysis of the DEGs was conducted. To confirm the reliability of the microarray results, the DEGs that showed the largest differences compared with non-stimulated GFs were further analyzed by RT-PCR. To evaluate the effect of polarization on GFs responses to lipopolysaccharide (LPS), GFs stimulated by 4 cytokines were further stimulated with Escherichia coli LPS and mRNA levels of several genes were analyzed using RT-PCR. Differentially expressed genes by 4Th cytokines were enriched in different GO terms, and the patterns of gene expression on GFs were shown functionally different. GFs stimulated with IFN-γ (GF(IFN-γ)) up-regulated the expression of chemokines (chemokine (C-X-C motif) ligand (CXCL)9, -10, -11, chemokine (C-C motif) ligand (CCL)8), molecules involved in antigen presentation, complement component 3 (C3), and other immune response-related molecules, whereas they down-regulated the expression of several types of collagen, extracellular matrix (ECM) components, and DNA replication and nuclear protein-related molecules. By contrast, GF(IL-4) up-regulated the expression of ECM components, cell adhesion molecules, and tissue development-related molecules and down-regulated the expression of chemokines (CXCL10 and CXCL8) and adaptive immune response-related molecules. GF(IL-17) up-regulated the expression of chemokines and other molecules for neutrophil infiltration and activation, the pro-inflammatory cytokine IL-6, and C3. GF(TGF-β) up-regulated the expression of cell growth-related molecules, ECM components, several types of collagen, and cell adhesion molecules and down-regulated the expression of molecules related to complement activation and bacterial recognition. GFs stimulated by 4 cytokines responded differently to LPS. These results show that Th cytokines can polarize GFs into cells with functionally distinct features: immune-activating but tissue-destructive GF(IFN-γ), tissue-reparative, and immune-inhibiting GF(IL-4), highly pro-inflammatory GF(IL-17), and potent tissue-reparative GF(TGF-β).

Comprehensive RNA-seq analysis revealed gene network associated with leaf morphogenesis in Vitis vinifera

ABSTRACT Leaf morphogenesis in Vitis vinifera is closely associated with the biosynthesis of various metabolites, starch, and growth-related molecules. In the present study, Illumina RNA deep sequencing-based on transcriptome analysis was deployed to understand leaf morphogenesis in Vitis vinifera. The KEGG pathway analysis revealed the transcripts involved in the biosynthesis of major metabolites leading to leaf growth. The highest number of DEGs were down-regulated at young leave-vs-mature leaves stages (3,501) followed by large leaves-vs-mature leaves (2,364); however, the minimum number of up-regulated transcripts were recorded at young leaves-vs-immature leaves (195). The gene regulation in the starch biosynthetic pathway, young leaves-vs-mature leaves showed the highest number of transcripts (24 up-regulated and 60 down-regulated). For ABC Transporters, 224 transcripts were expressed, among which the maximum number of transcripts (59) were recorded in large leaves-vs-mature leaves. Amino and nucleotide sugar metabolism showed 112 transcripts (22 up-regulated and 90 down-regulated), in which immature leaves-vs-mature leaves expressed 30 DEGs. In addition, Flavonoid pathway showed the highest number of transcripts i.e. 903 were expressed among the studied pathways. The findings of this study will also help to a better understanding of the underlying process during grape leaf morphogenesis and to unravel the mechanism.

RANKL Mediates Muscle Atrophy and Dysfunction in a Cigarette Smoke-induced Model of Chronic Obstructive Pulmonary Disease.

Skeletal muscle dysfunction is one of the important comorbidities of chronic obstructive pulmonary disease (COPD); however, the underlying mechanisms remain largely unknown. RANKL (receptor activator of nuclear factor κB ligand), a key mediator in osteoclast differentiation, was also found to play a role in skeletal muscle pathogenesis. Whether RANKL is involved in COPD-related skeletal muscle dysfunction is as-of-yet unknown. We examined the expression of RANKL/RANK in skeletal muscles from mice exposed to cigarette smoke (CS) for 24 weeks. Grip strength and exercise capacity as well as muscular morphology were evaluated in CS-exposed mice with or without anti-RANKL treatment. The expressions of protein synthesis- or muscle growth-related molecules (IGF-1, myogenin, and myostatin), muscle-specific ubiquitin E3 ligases (MuRF1 and atrogin-1), and the NF-κb inflammatory pathway were also evaluated in skeletal muscles. The effect of CS extract on RANKL/RANK expression and that of exogenous RANKL on the ubiquitin-proteasome pathway in C2C12 myotubes were investigated in vitro. Long-term CS exposure induced skeletal muscle dysfunction and atrophy together with upregulation of RANKL/RANK expression in a well-established mouse model of COPD. RANKL neutralization prevented skeletal muscle dysfunction and atrophy. RANKL inhibition decreased expressions of myostatin and MuRF1/Atrogin1 and suppressed the NF-κb pathway in skeletal muscles from CS-exposed mice. In in vitro experiments with C2C12 myotubes, CS extract induced expression of RANKL/RANK, and exogenous RANKL induced activation of the ubiquitin-proteasome pathway and NF-κb pathway via RANK. Our results revealed an important role of the RANKL/RANK pathway in muscle atrophy induced by CS exposure, suggesting that RANKL may be a potential therapeutic target in COPD-related skeletal muscle dysfunction.

NDAT Targets PI3K-Mediated PD-L1 Upregulation to Reduce Proliferation in Gefitinib-Resistant Colorectal Cancer.

The property of drug-resistance may attenuate clinical therapy in cancer cells, such as chemoresistance to gefitinib in colon cancer cells. In previous studies, overexpression of PD-L1 causes proliferation and metastasis in cancer cells; therefore, the PD-L1 pathway allows tumor cells to exert an adaptive resistance mechanism in vivo. Nano-diamino-tetrac (NDAT) has been shown to enhance the anti-proliferative effect induced by first-line chemotherapy in various types of cancer, including colorectal cancer (CRC). In this work, we attempted to explore whether NDAT could enhance the anti-proliferative effect of gefitinib in CRC and clarified the mechanism of their interaction. The MTT assay was utilized to detect a reduction in cell proliferation in four primary culture tumor cells treated with gefitinib or NDAT. The gene expression of PD-L1 and other tumor growth-related molecules were quantified by quantitative polymerase chain reaction (qPCR). Furthermore, the identification of PI3K and PD-L1 in treated CRC cells were detected by western blotting analysis. PD-L1 presentation in HCT116 xenograft tumors was characterized by specialized immunohistochemistry (IHC) and the hematoxylin and eosin stain (H&E stain). The correlations between the change in PD-L1 expression and tumorigenic characteristics were also analyzed. (3) The PD-L1 was highly expressed in Colo_160224 rather than in the other three primary CRC cells and HCT-116 cells. Moreover, the PD-L1 expression was decreased by gefitinib (1 µM and 10 µM) in two cells (Colo_150624 and 160426), but 10 µM gefitinib stimulated PD-L1 expression in gefitinib-resistant primary CRC Colo_160224 cells. Inactivated PI3K reduced PD-L1 expression and proliferation in CRC Colo_160224 cells. Gefitinib didn’t inhibit PD-L1 expression and PI3K activation in gefitinib-resistant Colo_160224 cells. However, NDAT inhibited PI3K activation as well as PD-L1 accumulation in gefitinib-resistant Colo_160224 cells. The combined treatment of NDAT and gefitinib inhibited pPI3K and PD-L1 expression and cell proliferation. Additionally, NDAT reduced PD-L1 accumulation and tumor growth in the HCT116 (K-RAS mutant) xenograft experiment. (4) Gefitinib might suppress PD-L1 expression but did not inhibit proliferation through PI3K in gefitinib-resistant primary CRC cells. However, NDAT not only down-regulated PD-L1 expression via blocking PI3K activation but also inhibited cell proliferation in gefitinib-resistant CRCs.

Korean red ginseng improves testicular ineffectiveness in aging rats by modulating spermatogenesis-related molecules

Korean red ginseng (Panax ginseng Meyer) is known to rejuvenate testicular effectiveness and the sperm maturation process by regulating redox proteins in aged rats. This study was performed to investigate the effect of Korean red ginseng water extract (KRG-WE) on the expression level of spermatogenesis-related key biomolecules and sex hormone receptors as well as enzymes regulating oxidation, histone deacetylation, and growth-related activities in aged rat testis. KRG-WE (200mg/kg) mixed with a regular pellet diet was administered to 12-month-old rats for 6months (KRG-AC), whereas the young (YC, 2months) and aged (AC, 12months) controls received the vehicle only. The results showed that the expression levels of spermatogenesis-related key biomolecules (inhibin-α, nectin-2, and cyclic adenosine monophosphate [cAMP] responsive element binding protein [CREB]-1), sex hormone receptors (androgen, luteinizing- and follicle-stimulating hormone receptors [AR, LHR, and FSHR, respectively]), and antioxidant enzymes (glutathione S-transferase mu [GSTm]-5, glutathione peroxidase [GPx]-4, peroxiredoxin [PRx]-3), as well as histone deactylation (silent mating type information regulation 2 homolog 1, SIRT1) and growth-related (mammalian target of rapamycin complex 1, mTORC1) molecules were significantly altered in the AC group rat testes compared with those of the YC group. However, KRG-WE treatment of the AC group significantly (p<0.05) attenuated these molecular changes. From these results, it can be concluded that long-term administration of KRG-WE significantly delayed the aging-induced testicular dysfunction.

Selective growth inhibition by suppression of F1Fo ATPase in caninemalignant melanoma cell lines.

Canine malignant melanoma (CMM) is a highly aggressive and fatal neoplasm. To identify potential therapeutic compounds and/or targets, 320 compounds were screened for their growth inhibitory activity in a CMM line (CMM-1) using a chemical library known to target specific signaling pathways/cell growth-related molecules. Among the compounds screened, the F1Fo ATPase inhibitor oligomycin showed potent growth inhibitory effects in CMM-1 cells, while exhibiting less toxic effects in a non-neoplastic control cell line (MDCK cells). The growth inhibitory effect of oligomycin A was then examined using six CMM lines and MDCK cells. Three CMM lines were highly sensitive to oligomycin A, with around 3000-20000 times lower IC50 compared with oligomycin A-resistant CMM lines and MDCK cells. Oligomycin A-sensitive CMM-1 cells exhibited much greater oligomycin A-induced decreases in cellular ATP compared to oligomycin A-resistant cell lines. Although the oligomycins are clinically unsuitable because of its invivo toxicity, these findings implicate the potential of F1Fo ATPase as a therapeutic target in a subset of CMM.

Exclusion of integrins from CNS axons is regulated by Arf6 activation and the AIS.

Integrins are adhesion and survival molecules involved in axon growth during CNS development, as well as axon regeneration after injury in the peripheral nervous system (PNS). Adult CNS axons do not regenerate after injury, partly due to a low intrinsic growth capacity. We have previously studied the role of integrins in axon growth in PNS axons; in the present study, we investigate whether integrin mechanisms involved in PNS regeneration may be altered or lacking from mature CNS axons by studying maturing CNS neurons in vitro. In rat cortical neurons, we find that integrins are present in axons during initial growth but later become restricted to the somato-dendritic domain. We investigated how this occurs and whether it can be altered to enhance axonal growth potential. We find a developmental change in integrin trafficking; transport becomes predominantly retrograde throughout axons, but not dendrites, as neurons mature. The directionality of transport is controlled through the activation state of ARF6, with developmental upregulation of the ARF6 GEF ARNO enhancing retrograde transport. Lowering ARF6 activity in mature neurons restores anterograde integrin flow, allows transport into axons, and increases axon growth. In addition, we found that the axon initial segment is partly responsible for exclusion of integrins and removal of this structure allows integrins into axons. Changing posttranslational modifications of tubulin with taxol also allows integrins into the proximal axon. The experiments suggest that the developmental loss of regenerative ability in CNS axons is due to exclusion of growth-related molecules due to changes in trafficking.

Effects of subacute hypothyroidism on metabolism and growth-related molecules.

Thyroid hormones are crucial hormones that primarily regulate the metabolism of entire body cells. In this study, Sprague-Dawley rats were grouped into sham thyroidectomy (Sham Tx), thyroidectomy (Tx), Tx with thyroxine replacement (Tx + T4), and PTU injection (PTU) groups. Metabolic parameters were measured by means of metabolic cages for 14 days. After 14 days, the rats were sacrificed while the levels of plasma or serum TSH and growth-related molecules, such as active and total ghrelin, GH, and IGF-1, were assayed. The results revealed that hypothyroid rats tended to eat less food and experienced substantial body weight gain, whereas the rats with T4 replacement tended to eat more food while continuing to lose weight. In hypothyroid rats, the growth-related molecules, such as active ghrelin and total ghrelin secretion, were enhanced, and the ghrelin receptors were also up-regulated. However, circulating GH levels were not elevated and IGF-1 secretion was inhibited in hypothyroid rats. In the Tx + T4 group, the changes of active ghrelin, total ghrelin, GHS-R expression, and IGF-1 were reversed, whereas the GH secretion was higher than that of the Sham Tx group and hypothyroid groups. This study resulted in the novel finding that the ghrelin/GHS-R axis and GH/IGF-1 axis are interrupted in hypothyroid rats.

Abstract 2771: HSP90 inhibitor NVP-AUY922 exhibits the potent antiproliferative effect in gastrointestinal cancer cells with a lack of PTEN expression

Abstract Background: The heat shock protein 90 (HSP90), a molecular chaperone, has provoked great interest as a promising molecular target for cancer treatment, since it is involved in regulating the conformation, stability and functions of key oncogenic proteins. At present, a variety of chemical compounds targeting HSP90 have been developed and have shown convincing anti-neoplastic activity in various preclinical tumor models. Several inhibitors of HSP90 have been reported exhibiting more antitumor efficacy in PTEN-null tumor xenograft. The aim of our study was to evaluate the antitumor effects of a novel HSP90 inhibitor, NVP-AUY922, in human malignant cells, especially in PTEN-null cancer cells. Materials and Methods: Human esophageal cancer (TE-1, TE-4, TE-8, TE-10), gastric cancer (NUGC-3, NUGC-3/IL), pancreatic cancer (PANC-1, MIAPACA-2, Bxpc3) and mutant c-kit-driven lymphocytes (Ba/F3-KE11, Ba/F3-K642, Ba/F3-820Mz), mimicking gastrointestinal stromal tumor cells, were examined. WST assay and/or trypan blue exclusion assay were conducted to assess the anti-proliferation effects of NVP-AUY922 in those cells mentioned above. The activity of cell growth-related molecules such as AKT, ERK, and PTEN were investigated by western blot analysis. Genetic modification using siRNA and expression vector were applied for silencing and overexpression of PTEN, respectively, in cancer cells. Results: Cell viability assays showed that NVP-AUY922 potently inhibits the proliferation of human cancer cell lines, regardless of 5-FU resistance or c-Kit mutation, by which the sensitivity to imatinib can be affected. PTEN-null TE-4 cells exhibited more sensitivity to NVP-AUY922. Interestingly, The sensitivity to NVP-AUY922 was increased by silencing PTEN expression in intact PTEN-expressing TE-10 cells and decreased by exogenous transduction of PTEN in TE-4 cells. Furthermore, NVP-AUY922 significantly inhibited the activity of AKT and ERK in TE-4 cells, but not in TE-10 cells. Conclusion: Inhibition of Hsp90 function by NVP-AUY922 exhibited strong cytotoxicity against most of the cancer cells, including some anticancer drug-resistant cells, revealing its potential for a novel therapeutic alternative to cancer treatment. In addition, this study suggests that the expression status of PTEN determines the sensitivity to HSP90 inhibitor for cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2771. doi:1538-7445.AM2012-2771

Effects of two monoclonal antibodies, MLS128 against Tn-antigen and 1H7 against insulin-like growth factor-I receptor, on the growth of colon cancer cells

MLS128 is an anti-carbohydrate monoclonal antibody (mAb) that binds three or two consecutive Tn-antigens. MLS128 bound 110-210 kDa glycoproteins (GPs) and inhibited the growth of LS180 and HT29 colon and MCF-7 breast cancer cells. One possible mechanism of MLS128's inhibition of growth may be via insulin-like growth factor-I receptor (IGF-IR) down-regulation (Morita et al. BioScience Trends. 2009; 3:32-37). The current study examined the role of IGF-IR signaling in the growth of colon cancer cells and its possible interaction with MLS128-induced inhibition of cell growth in LS180, LS174T, and HT29 human colon cancer cells treated with MLS128 or anti-IGF-IR 1H7. Both MLS128 and 1H7 treatment significantly inhibited the growth of colon cancer cells. All three colon cancer cell lines expressed IGF-IR. Their growth was in part IGF-I dependent, but inhibition by MLS128 was independent of IGF-IR signaling. All of the colon cancer cell lines expressed an 110kDa GP for MLS128 binding, but MCF-7 cells expressed MLS128-detectable bands with higher molecular masses. 1H7 treatments caused down-regulation of IGF-IR but did not affect 110kDa GP levels. MLS128 treatments resulted in partial disappearance of the 110kDa band but did not affect IGF-IR levels. Western blotting analyses of colon and breast cancer cell lysates revealed that colon and breast cancer cells differed significantly in patterns of expression of growth-related molecules while colon cancer cells were similar but distinctive. In conclusion, MLS128 inhibited the growth of colon cancer cells by binding to the 110kDa GP receptor. Inhibition of growth by MLS128 did not appear to affect IGF-IR signaling and instead only affected other growth signaling pathways.

Myeloid-derived suppressor cells exhibit two bioenergetic steady-states in vitro

Growing tumours have acquired several mechanisms to resist to immune recognition. Among these strategies, myeloid-derived suppressor cells (MDSCs) contribute to tumour escape by suppressing T-cell specific anti-tumoural functions. The development of therapies that could specifically inhibit MDSC maturation, recruitment, accumulation and immunosuppressive functions is thus of great interest. This requires the identification of valuable biomarkers of MDSC behaviour in vitro. As for immune cells, whose energetic state is known as a biomarker of their functionality, we have characterized in vitro the metabolic and energetic behaviour of MSC-1 cells, an immortalized cell line derived from mouse MDSCs and used as model cell line. Combined results from in vitro 31P-NMR with living cells and HPLC–MS analyses from cell extracts allowed to identify two distinct bioenergetic steady-states that coincided with exponential and stationary growth phases. While the adenylate energy charge remained constant throughout the culture duration, both the percentage of total pyrimidines, the UTP-to-ATP and PME (phosphomonoesters)-to-NTP ratios were higher at the exponential growth phase compared to the plateau phase, suggesting metabolically active cells and the production of growth-related molecules. Conversely, the NTP ratio increased at the entry of the stationary phase revealing the deterioration of the global bioenergetic status and the arrest of anabolic processes.

The Effect of Helicobacter Pylori and Economic Status on Growth Parameters and Leptin, Ghrelin, and Insulin-Like Growth Factor (IGF)-I Concentrations in Children

It was suggested that gastric colonization with Helicobacter pylori (H. pylori) was associated with suboptimal nutrition and growth in childhood. Furthermore, several studies indicated a relationship between H. pylori colonization and alterations in the circulating levels of growth-related molecules (GRM). Accordingly, in this study, we investigate the effect of H. pylori infection on GRMs and on the growth of healthy school children, taking into consideration the effect of their economic status (ES) and anthropometric indices of their parents. To acquire sociodemographic and anthropometric nutritional parameters and to detect H. pylori-specific serum IgG antibodies and growth-related molecules, we evaluated a total of 473 children attending four different primary and secondary schools in Istanbul. Subsequently, we assessed the effect of H. pylori on growth-related parameters (weight for age SDS, height for age SDS, BMI SDS, TSF, and waist-to-hip ratio) and on GRMs (leptin, ghrelin, and insulin-like growth factor-1 (IGF-1)), controlling for age, gender, family income, household crowding (HC), breastfeeding, maternal and paternal BMI SDS, and midparental height SDS with complex statistical models. Of the 473 children (275 F/198 M, age 6-15 years; mean: 10.3 ± 0.1 years), 161 (34%) were H. pylori-positive. The prevalence of H. pylori was significantly higher in lower economic status (ES) groups, in children living in crowded houses, and in older age groups. Using simple statistical models, we did not find any significant associations between H. pylori infection and the growth parameters. However, in complex models for height for age SDS and for weight for age SDS, there was a significant interaction between H. pylori infection status and ES. Whereas in H. pylori-positive subjects, mid-income family children were both taller and heavier than the low-income group, there was no such an association in H. pylori-negative subjects. Among biochemical parameters, only ghrelin levels were associated with H. pylori infection in all models. Leptin levels were associated with HC in girls, whereas none of the parameters was significantly associated with leptin levels in boys. For IGF-1 levels, for boys, age and maternal BMI, and for girls, age and HC were significantly associated with IGF-1 levels. We suggest that H. pylori may impair growth significantly only in susceptible children where unfavorable socioeconomic conditions facilitate its action, probably through mechanisms, at least in part, involving growth-related molecules.

Involvement of ERK1/2 signalling and growth-related molecules’ expression in response to heat stress-induced damage in rat jejunum and IEC-6 cells

Our previous studies found small intestine epithelial tissues from several different animals (including rats, pigs and chickens) became significantly damaged following exposure to extreme heat. However, damaged tissue was rapidly repaired or regenerated in the following few days. Growth-related molecules are critical for cellular survival and promote endothelial cell proliferation and migration. The ERK1/2 signalling pathway is reported to regulate the growth and adaptation of endothelial cells to both physiological and pathological stimuli. However, little information is available concerning both growth-related molecules and ERK1/2 in response to heat stress. Herein, we employed both live rats and rat IEC-6 cells to investigate growth-related molecule expression and ERK1/2 activation in heat stress. Heat stress caused significant morphological damage to rat intestinal tissue and IEC-6 cells, reduced cell growth and proliferation, induced apoptosis, altered growth-related molecule mRNA expression and increased ERK1/2 phosphorylation. Addition of U0126 (a selective inhibitor of MEK kinase responsible for ERK phosphorylation) combined with heat stress exacerbated the morphological damage and apoptosis. With the addition of U0126, further up- or down-regulation of Egfr, Ctgf, Tgif, Vegfa, Okl38 and Gdf15 in response to heat stress was observed. In conclusion, extreme heat stress caused obvious damage to rat jejunum and IEC-6 cells. Both growth-related molecule expression and ERK1/2 phosphorylation were involved in response to heat stress. ERK1/2 inhibition exacerbated apoptosis and affected growth factor mRNA expression in heat stress.

Irradiated fibroblast‐induced bystander effects on invasive growth of squamous cell carcinoma under cancer–stromal cell interaction

The irradiated fibroblast-induced response of non-irradiated neighboring cells is called 'radiation-induced bystander effect', but it is unclear in non-irradiated human squamous cell carcinoma (SCC) cells. The present study shows that irradiated fibroblasts promoted the invasive growth of T3M-1 SCC cells, but not their apoptosis, more greatly than non-irradiated fibroblasts, using collagen gel invasion assay, immunohistochemistry and Western blot. The number of irradiated fibroblasts decreased to about 30% of that of non-irradiated fibroblasts, but irradiated fibroblasts increased the growth marker ki-67 display of SCC cells more greatly than non-irradiated fibroblasts. Irradiated fibroblasts did not affect the apoptosis marker ss-DNA expression of SCC cells. Irradiated fibroblasts enhanced the display of the following growth-, invasion- and motility-related molecules in SCC cells more greatly than non-irradiated fibroblasts: c-Met, Ras, mitogen-activated protein kinase (MAPK) cascade (Raf-1, MEK-1 and ERK-1/2), matrix metalloproteinase-1 and -9, laminin 5 and filamin A. Irradiated fibroblasts, but not non-irradiated ones, formed irradiation-induced foci (IRIF) of the genomic instability marker p53-binding protein 1 (53BP1) and expressed transforming growth factor-beta1 (TGF- beta1). Irradiated fibroblasts in turn enabled SCC cells to enhance 53BP1 IRIF formation more extensively than non-irradiated fibroblasts. Finally, effects of irradiated fibroblasts on growth and apoptosis of another HEp-2 SCC cell type were similar to those of T3M-1. These results suggest that irradiated fibroblasts promotes invasion and growth of SCC cells by enhancement of invasive growth-related molecules above through TGF- beta1-mediated bystander mechanism, in which irradiated fibroblast-induced genomic instability of SCC cells may be involved.

Comparative proteome analysis of rat brain and coronary microvascular endothelial cells

The endothelium of different organs displays a remarkable heterogeneity, although it presents many common functional and morphological features. However, despite our knowledge of heterogeneity among endothelial cells from different sites, the differences between brain microvascular endothelial cells (BMEC) and coronary microvascular endothelial cells (CMEC) are poorly defined. The aim of this study was to investigate whether BMEC are distinct from CMEC at the protein level. Using the proteomic approach, we comparatively analyzed the proteome of cultured BMEC and CMEC. We reproducibly separated over 2000 polypeptides by using two-dimensional electrophoresis (2-DE) at pH range of 3-10. Using PDQuest software to process the 2-DE gel images, forty-seven protein spots were differentially expressed in the two-endothelial cells. Of these, thirty-five proteins are highly expressed in BMEC, whereas twelve proteins are highly expressed in CMEC. Fifteen proteins in BMEC and seven proteins in CMEC were identified with high confidence by matrix-associated laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). Our data suggested that BMEC and CMEC were different in several aspects including cytokine and growth-related molecules, stress-related proteins, metabolic enzymes, signal transduction proteins and others. The identification of a set of proteins preferentially expressed in BMEC and CMEC provided new data on the heterogeneity of the endothelium.

Differences in the regenerative response of neuronal cell populations and indications for plasticity in intraspinal neurons after spinal cord transection in adult zebrafish

In zebrafish, the capacity to regenerate long axons varies among different populations of axotomized neurons after spinal cord transection. In specific brain nuclei, 84–92% of axotomized neurons upregulate expression of the growth-related genes GAP-43 and L1.1 and 32–51% of these neurons regrow their descending axons. In contrast, 16–31% of spinal neurons with axons ascending to the brainstem upregulate these genes and only 2–4% regrow their axons. Dorsal root ganglion (DRG) neurons were not observed to regrow their ascending axons or to increase expression of GAP-43 mRNA. Expression of L1.1 mRNA is high in unlesioned and axotomized DRG neurons. In the lesioned spinal cord, expression of growth-related molecules is increased in a substantial population of non-axotomized neurons, suggesting morphological plasticity in the spinal-intrinsic circuitry. We propose that locomotor recovery in spinal-transected adult zebrafish is influenced less by recovery of ascending pathways, but more by regrowth of descending tracts and rearrangement of intraspinal circuitry.

Temporal and spatial distribution of growth‐associated molecules and astroglial cells in the rat corticospinal tract during development

To understand better the role of growth-promoting and -inhibiting molecules in the development of the corticospinal tract (CST), temporospatial expression of embryonic neural cell adhesion molecule (E-NCAM), growth-associated protein-43 (GAP-43), and chondroitin sulfate proteoglycan (CSPG) was studied in developing rats. Transverse sections of the seventh cervical (C7), seventh thoracic (T7), and fourth lumbar (L4) segments were examined at postnatal days (P) 2, 6, 10, 14, and 28. The highest E-NCAM immunoreactivity appeared at the C7 level on P2 and shifted caudally to the T7 on P6 and L4 on P10, which correlated closely with the time course of CST development. The peak expression of GAP-43 emerged at C7 on P2 and shifted to the T7 and L4 levels at a relatively lagging pace compared with that of E-NCAM. Conversely, a transient reduction in CSPG immunoreactivity was found within the CST at the C7 level on P2, T7 level on P6, and L4 level on P10, corresponding well with the arrival of CST-leading axons at these levels. Interestingly, higher levels of CSPG were found to surround the growing CST, suggesting a repulsive environment that channels the growth of CST. Moreover, a transition from immature to mature astrocytes in a rostrocaudal direction during CST development was evidenced by anti-vimentin and anti-glial fibrillary acidic protein (GFAP) immunostaining, suggesting a guidance role of immature astroglia in axonal outgrowth. Our study thus demonstrated dynamic changes of multiple growth-related molecules and astroglial environment that contribute to postnatal development of the CST.

Air–liquid interface promotes invasive growth of laryngeal squamous cell carcinoma with or without hypoxia

Air–liquid interface (ALI) is a microenvironment of aerodigestive tract. In our previous study, ALI promoted invasive growth of laryngeal squamous cell carcinoma (SCC); but its mechanism was unclear. Hypoxia is also related to cancer spread. Here we show that ALI with or without hypoxia accelerated invasive growth of laryngeal SCC cells, using collagen gel invasion assay. Submerged condition (SMC) without ALI did not induce the invasion with or without hypoxia. ALI enhanced expression of the following growth-, invasion-, and motility-related molecules in the cells with or without hypoxia more greatly than SMC: c-Met, Ras, mitogen-activated protein kinase cascade proteins (Raf-1, MEK-1, and ERK-1/2), matrix metalloproteinase-1, and filamin A. The data indicate that ALI promotes invasive growth of SCC cells by enhancement of the invasive growth-related molecules above, through mechanisms that differ from hypoxia, suggesting that ALI microenvironment should be taken into account for the study of cancer biology.

Dynamics of gene expression for immediate early- and late genes after seizure activity in aged rats

The ability of the rodent brain to support plasticity-related phenomena declines with increasing age. A decreased coordination of genes implicated in brain plasticity may be one factor contributing to this decline. Synaptic rearrangement that occurs after seizure activity is regarded as a model of brain plasticity. In a rat model of seizure-related brain plasticity, we found that the induction of immediate-early genes, as exemplified by c- fos and tissue plasminogen activator ( tPA), is not impaired in the aged rat brain. However, the aged rat brain responded more slowly to chemically induced seizure, and the levels of c- fos and tPA mRNAs induction are decreased in the cortex and in the hippocampus of 30 month old rats, as compared to the levels expressed by 3 month old rats. In addition, at the peak induction, the TPA transcripts were restricted to certain cortical layers of the older rats. Surprisingly, in applying the same experimental paradigm to late genes, we found that there was a shift toward earlier times in the maximum expression of growth-related molecules, the microtubule-associated protein 1B (MAP1B) mRNA, which was very evident in 18 month old rats. Aberrant immunolabeling of MAP1B occurred in cortical layer VI of the aged rats where, unlike in young rats, there was heavy staining of neuronal somata. These results suggest that (1) one consequence of aging, besides decreases in the levels of mRNA, is a progressive loss of coordination in gene activity following the administration of a stimulus; (2) since c-fos, TPA and MAP1B have been implicated in neuronal plasticity, these findings could explain, in part, the limited plasticity of the aging brain.

Brain plasticity: to what extent do aged animals retain the capacity to coordinate gene activity in response to acute challenges.

The ability of the rodent brain to support plasticity-related phenomena declines with increasing age. A decreased coordination of genes implicated in brain plasticity may be one factor contributing to this decline. Synaptic rearrangement that occurs after seizure activity is regarded as a model of brain plasticity. In a rat model of seizure-related brain plasticity, we found that the induction of immediate-early genes, as exemplified by c-fos and tissue plasminogen activator (TPA) is not impaired in the aged rat brain. However, the aged rat brain responded more slowly to chemically induced seizure and the levels of c-fos and TPA mRNAs induction are decreased in the cortex and in the hippocampus of 30-month-old rats, as compared to the levels expressed by 3-month-old rats. In addition, at the peak induction the TPA transcripts were restricted to certain cortical layers of the older rats. Surprisingly, in applying the same experimental paradigm to late genes we found that there was a shift toward earlier times in the maximum expression of growth-related molecule, the microtubule-associated protein 1B (MAP1B) mRNA, which was very evident in 18-month-old rats. Aberrant immunolabeling of MAP1B occurred in cortical layer VI of the aged rats where, unlike in young rats, there was heavy staining of neuronal somata. These results suggest that (i) one consequence of aging, besides decreases in the levels of mRNA, is a progressive loss of coordination in gene activity following the administration of a stimulus; (ii) since c-fos, TPA and MAP1B have been implicated in neuronal plasticity, these findings could explain, in part, the limited plasticity of the aging brain.