Transcriptional changes in U343 MG-a glioblastoma cell line exposed to ionizing radiation

Glioblastoma multiforme (GBM) is a highly invasive and radioresistant brain tumor. Aiming to study how glioma cells respond to gamma-rays in terms of biological processes involved in cellular responses, we performed experiments at cellular context and gene expression analysis in U343-MG-a GBM cells irradiated with 1 Gy and collected at 6 h post-irradiation. The survival rate was approximately 61% for 1 Gy and was completely reduced at 16 Gy. By performing the microarray technique, 859 cDNA clones were analyzed. The Significance Analysis of Microarray algorithm indicated 196 significant expressed genes (false discovery rate (FDR) = 0.42%): 67 down-regulated and 97 up-regulated genes, which belong to several classes: metabolism, adhesion/cytoskeleton, signal transduction, cell cycle/apoptosis, membrane transport, DNA repair/DNA damage signaling, transcription factor, intracellular signaling, and RNA processing. Differential expression patterns of five selected genes (HSPA9B, INPP5A, PIP5K1A, FANCG, and TPP2) observed by the microarray analysis were further confirmed by the quantitative real time RT-PCR method, which demonstrated an up-regulation status of those genes. These results indicate a broad spectrum of biological processes (which may reflect the radio-resistance of U343 cells) that were altered in irradiated glioma cells, so as to guarantee cell survival.

The Protective Effect of CAPE on Hepatic Ischemia/Reperfusion Injury in Rats

Background/Aims. The transcription factor nuclear factor-kappa B (NF-kappa B) exerts a pivotal role in the pathogenesis of hepatic ischemia/reperfusion (I/R) injury. Caffeic acid phenyl ester (CAPE), a potent and specific NF-kappa B inhibitor, presents protective effects on I/R injury in some tissues. This study aimed to evaluate the effect of CAPE on hepatic I/R injury in rats. Materials and methods. Wistar rats were submitted to a sham operation, 60 min ischemia, or 60 min ischemia plus saline or CAPE treatment followed by 6 h reperfusion. Liver tissue injury was evaluated by alanine aminotransferase, aspartate aminotransferase, and tissue glutathione measurement, and histological damage score. Apoptotic hepatocytes were determined by the transferase-mediated dUTP-biotin nick-end labeling assay. Hepatic neutrophil accumulation was assessed by the naphthol method. Lipid peroxidation and NF-kappa B activation were evaluated by 4-hydroxynonenal and NF-kappa B p65 immunohistochemistry, respectively. Results. Animals submitted to ischemia showed a marked increase of alanine aminotransferase and aspartate aminotransferase after reperfusion, but with lower levels in CAPE group. Tissue glutathione content declined gradually during ischemia to reperfusion and was partially recovered with CAPE treatment. The histological damage score, apoptosis index, and neutrophil infiltration, as well as 4-hydroxynonenal and NF-kappa B p65 nuclear labeling, were higher in the liver of animals submitted to I/R compared to the ischemia group. However, the CAPE treatment significantly reduced all of these alterations. Conclusions. CAPE was able to protect the liver against normothermic I/R injury in rats. This effect may be associated with the inhibition of the NF-kappa B signaling pathway and decrease of the acute inflammatory response following I/R in the liver. (C) 2008 Elsevier Inc. All rights reserved.

Identification of a myeloid committed progenitor as the cancer-initiating cell in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by a block in differentiation and accumulation of promyelocytes in the bone marrow and blood. The majority of APL patients harbor the t(15: 17) translocation leading to expression of the fusion protein promyelocytic-retinoic acid receptor alpha. Treatment with retinoic acid leads to degradation of promyelocytic-retinoic acid receptor alpha protein and disappearance of leukemic cells; however, 30% of APL patients relapse after treatment. One potential mechanism for relapse is the persistence of cancer ""stem"" cells in hematopoietic organs after treatment. Using a novel sorting strategy we developed to isolate murine myeloid cells at distinct stages of differentiation, we identified a population of committed myeloid cells (CD34(+), c-kit(+), Fc gamma RIII/II(+), Gr1(int)) that accumulates in the spleen and bone marrow in a murine model of APL. We observed that these cells are capable of efficiently generating leukemia in recipient mice, demonstrating that this population represents the APL cancer-initiating cell. These cells down-regulate the transcription factor CCAAT/enhancer binding protein alpha (C/EBP alpha) possibly through a methylation-dependent mechanism, indicating that C/EBP alpha deregulation contributes to transformation of APL cancer-initiating cells. Our findings provide further understanding of the biology of APL by demonstrating that a committed transformed progenitor can initiate and propagate the disease. (Blood. 2009; 114: 5415-5425)

Evidence for a network transcriptional control of promiscuous gene expression in medullary thymic epithelial cells

The expression of peripheral tissue antigens (PTAs) in the thymus by medullary thymic epithelial cells (mTECs) is essential for the central self-tolerance in the generation of the T cell repertoire. Due to heterogeneity of autoantigen representation, this phenomenon has been termed promiscuous gene expression (PGE), in which the autoimmune regulator (Aire) gene plays a key role as a transcription factor in part of these genes. Here we used a microarray strategy to access PGE in cultured murine CD80(+) 3.10 mTEC line. Hierarchical clustering of the data allowed observation that PTA genes were differentially expressed being possible to found their respective induced or repressed mRNAs. To further investigate the control of PGE, we tested the hypothesis that genes involved in this phenomenon might also be modulated by transcriptional network. We then reconstructed such network based on the microarray expression data, featuring the guanylate cyclase 2d (Gucy2d) gene as a main node. In such condition, we established 167 positive and negative interactions with downstream PTA genes. Silencing Aire by RNA interference, Gucy2d while down regulated established a larger number (355) of interactions with PTA genes. T- and G-boxes corresponding to AIRE protein binding sites located upstream to ATG codon of Gucy2d supports this effect. These findings provide evidence that Aire plays a role in association with Gucy2d, which is connected to Several PTA genes and establishes a cascade-like transcriptional control of promiscuous gene expression in mTEC cells. (C) 2009 Elsevier Ltd. All rights reserved.

Factors involved in the T helper type 1 and type 2 cell commitment and osteoclast regulation in inflammatory apical diseases

Periapical chronic lesion formation involves activation of the immune response and alveolar bone resorption around the tooth apex. However, the overall roles of T helper type 1 (Th1), Th2, and T-regulatory cell (Treg) responses and osteoclast regulatory factors in periapical cysts and granulomas have not been fully determined. This study aimed to investigate whether different forms of apical periodontitis, namely cysts and granulomas, show different balances of Th1, Th2 regulators, Treg markers, and factors involved in osteoclast chemotaxis and activation. Gene expression of these factors was assessed using quantitative real-time polymerase chain reaction, in samples obtained from healthy gingiva (n = 8), periapical granulomas (n = 20), and cysts (n = 10). Periapical cysts exhibited a greater expression of GATA-3, while a greater expression of T-bet, Foxp3, and interleukin-10 (IL-10) was seen in granulomas. The expression of interferon-gamma, IL-4, and transforming growth factor-beta was similar in both lesions. Regarding osteoclastic factors, while the expression of SDF-1 alpha/CXCL12 and CCR1 was higher in cysts, the expression of RANKL was significantly higher in granulomas. Both lesions exhibited similar expression of CXCR4, CK beta 8/CCL23, and osteoprotegerin, which were significantly higher than in control. Our results showed a predominance of osteoclast activity in granulomas that was correlated with the Th1 response. The concomitant expression of Treg cell markers suggests a possible suppression of the Th1 response in granulomas. On the other hand, in cysts the Th2 activity is augmented. The mechanisms of periradicular lesion development are still not fully understood but the imbalance of immune and osteoclastic cell activity in cysts and granulomas seems to be critically regulated by Treg cells.

Role of NFKB2 on the early myeloid differentiation of CD34+ hematopoietic stem/progenitor cells

To better understand the early events regulating lineage-specific hematopoietic differentiation, we analyzed the transcriptional profiles of CD34+ human hematopoietic stem and progenitor cells (HSPCs) subjected to differentiation stimulus. CD34+ cells were cultured for 12 and 40 h in liquid cultures with supplemented media favoring myeloid or erythroid commitment. Serial analysis of gene expression (SAGE) was employed to generate four independent libraries. By analyzing the differentially expressed regulated transcripts between the un-stimulated and the stimulated CD34+ cells, we observed a set of genes that was initially up-regulated at 12 h but were then down-regulated at 40 h, exclusively after myeloid stimulus. Among those we found transcripts for NFKB2, RELB, IL1B, LTB, LTBR, TNFRSF4, TGFB1, and IKBKA. Also, the inhibitor NFKBIA (IKBA) was more expressed at 12 h. All those transcripts code for signaling proteins of the nuclear factor kappaB pathway. NFKB2 is a subunit of the NF-kappa B transcription factor that with RELB mediates the non-canonical NF-kappa B pathway. Interference RNA (RNAi) against NFKB1, NFKB2 and control RNAi were transfected into bone marrow CD34+HSPC. The percentage and the size of the myeloid colonies derived from the CD34+ cells decreased after inhibition of NFKB2. Altogether, our results indicate that NFKB2 gene has a role in the early commitment of CD34+HSPC towards the myeloid lineage. (C) 2010 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.

Identification of genes differentially expressed in a strain of the mold Aspergillus nidulans carrying a loss-of-function mutation in the palA gene

To identify genes differentially expressed in a strain of the mold Aspergillus nidulans carrying a loss-of-function mutation in palA, a gene in the pH-responsive signal transduction pathway, suppression subtractive hybridization was performed between RNA isolated from the biA1 and biA1 palA1 strains grown under limiting inorganic phosphate at pH 5.0. We have identified several genes upregulated in the biA1 palA1 mutant strain that play important roles in mitotic fidelity, stress responses, enzyme secretion, signal transduction mechanisms, development, genome stability, phosphate sensing, and transcriptional regulation among others. The upregulation of eight of these transcripts was also validated by Northern blot. Moreover, we show that a loss of function mutation in the palA gene drastically reduced the neutral sugar content of the acid phosphatase PacA secreted by the fungus A. nidulans grown at pH 5.0 compared with a control strain.

Evaluating budesonide efficacy in nasal polyposis and predicting the resistance to treatment

Cell resistance to glucocorticoids is a major problem in the treatment of nasal polyposis (NP). The objectives of this study were to observe the effect of budesonide on the expression of IL-1 beta, TNF-alpha, granulocyte macrophage-colony stimulating factor, intercellular adhesion molecule (ICAM)-1, basic fibroblast growth factor, eotaxin-2, glucocorticoid receptor (GR)-alpha, GR-beta, c-Fos and p65 in nasal polyps and to correlate their expression to clinical response. Biopsies from nasal polyps were obtained from 20 patients before and after treatment with topical budesonide. Clinical response to treatment was monitored by a questionnaire and nasal endoscopy. The mRNA levels of the studied genes were measured by real-time quantitative (RQ)-PCR. There was a significant decrease in the expression of TNF-alpha (P < 0.05), eotaxin-2 (P < 0.05) and p65 (P < 0.05) in NP after treatment. Poor responders to glucocorticoids showed higher expression of IL-1 beta (3.74 vs. 0.14; P < 0.005), ICAM-1 (1.91 vs. 0.29; P < 0.05) and p65 (0.70 vs. 0.16; P < 0.05) before treatment. Following treatment, IL-1 beta (4.18 vs. 0.42; P < 0.005) and GR-beta (0.95 vs. 0.28; P < 0.05) mRNA expression was higher in this group. Topical budesonide reduced the expression of TNF-alpha, eotaxin-2 and p65. Poor responders to topical budesonide exhibit higher levels of IL-1 beta, ICAM-1 and nuclear factor (NF)-kappa B at diagnosis and higher expression of both IL-1 beta and GR-beta after treatment. These results emphasize the anti-inflammatory action of topical budesonide at the molecular level and its importance in the treatment of NP. Nevertheless, IL-1 beta, ICAM-1 and NF-kappa B may be associated with primary resistance to glucocorticoids in NP, whereas higher expression of GR-beta in poor responders only after glucocorticoid treatment may represent a secondary drug resistance mechanism in this disease.

Transcriptional profiling reveals genes in the human pathogen Trichophyton rubrum that are expressed in response to pH signaling

Trichophyton rubrum is a dermatophyte that infects human skin and nails. Its growth on keratin as its carbon source shifts the ambient pH from acidic to alkaline, which may be an efficient strategy for its successful infection and maintenance in the host. In this study, we used suppression subtractive hybridization to identify genes preferentially expressed in T rubrum incubated at either pH 5.0 or pH 8.0. The functional grouping of the 341 overexpressed unigenes indicated proteins putatively involved in diverse cellular processes, such as membrane remodeling, cellular transport, metabolism, cellular protection, fungal pathogenesis, gene regulation, interaction with the environment, and iron uptake. Although the basic metabolic machinery identified under both growth conditions seems to be functionally similar, distinct genes are upregulated at acidic or alkaline pHs. We also isolated a large number of genes of unknown function, probably unique to T rubrum or dermatophytes. Interestingly, the transcriptional profiling of several genes in a pacC mutant suggests that, in T rubrum, the transcription factor PacC has a diversity of metabolic functions, in response to either acidic or alkaline ambient pH. (C) 2009 Elsevier Ltd. All rights reserved.

Non-coding transcript in T cells (NTT): Antisense transcript activates PKR and NF-kappa B in human lymphocytes

T cell activation is a complex process involving many steps and the role played by the non-protein-coding RNAs (ncRNAs) in this phenomenon is still unclear. The non-coding T cells transcript (NTT) is differentially expressed during human T cells activation, but its function is unknown. Here, we detected a 426 m NTT transcript by RT-PCR using RNA of human lymphocytes activated with a synthetic peptide of HIV-1. After cloning, the sense and antisense 426 nt NTT transcripts were obtained by in vitro transcription and were sequenced. We found that both transcripts are highly structured and are able to activate PKR. A striking observation was that the antisense 426 nt NTT transcript is significantly more effective in activating PKR than the corresponding sense transcript. The transcription factor NF-kappa B is activated by PKR through phosphorylation and subsequent degradation of its inhibitor I-kappa B beta. We also found that the antisense 426 nt NTT transcript induces more efficiently the degradation Of I-kappa B beta than the sense transcript. Thus, this study suggests that the role played by NTT in the activation of lymphocytes can be mediated by PKR through NF-kappa B activation. However, the physiological significance of the activity of the antisense 426 nt NTT transcript remains unknown. (c) 2007 Elsevier Inc. All rights reserved.

Differential expression of HDAC3, HDAC7 and HDAC9 is associated with prognosis and survival in childhood acute lymphoblastic leukaemia

Altered expression of histone deacetylases (HDACs) is a common feature in several human malignancies and may represent an interesting target for cancer treatment, including haematological malignancies. We evaluated the mRNA gene expression profile of 12 HDAC genes by quantitative real-time polymerase chain reaction in 94 consecutive childhood acute lymphoblastic leukaemia (ALL) samples and its association with clinical/biological features and survival. ALL samples showed higher expression levels of HDAC2, HDAC3, HDAC8, HDAC6 and HDAC7 when compared to normal bone marrow samples. HDAC1 and HDAC4 showed high expression in T-ALL and HDAC5 was highly expressed in B-lineage ALL. Higher than median expression levels of HDAC3 were associated with a significantly lower 5-year event-free survival (EFS) in the overall group of patients (P = 0.03) and in T-ALL patients (P = 0.01). HDAC7 and HADC9 expression levels higher than median were associated with a lower 5-year EFS in the overall group (P = 0.04 and P = 0.003, respectively) and in B-lineage CD10-positive patients (P = 0.009 and P = 0.005, respectively). Our data suggest that higher expression of HDAC7 and HDAC9 is associated with poor prognosis in childhood ALL and could be promising therapeutic targets for the treatment of refractory childhood ALL.

WNT-5A, but not matrix metalloproteinase 3 or beta-catenin protein, expression is related to early stages of lip carcinogenesis

Background: Oncogenic Wnt/beta-catenin signaling occurs in numerous types of cancers, but little is known about the role of the Wnt protein family member, WNT-5A, in lip carcinogenesis. The aim of this study was to investigate WNT-5A, beta-catenin, and matrix metalloproteinase (MMP)-3 protein expression in actinic cheilitis (AC), and lip squamous cell carcinoma (LSCC). Methods: Twenty-one cases of AC, and fifty-one cases of LSCC were analyzed, with normal lip mucosa used as a control. Qualitative and semi-quantitative analyses of WNT-5A, beta-catenin, and MMP-3 immunostaining pattern and cellular distribution were performed. Results: WNT-5A was observed in more than 50% of the cells, scattered in all layers of AC, in contrast to the absence of immunostaining in normal lip mucosa. AC presented a higher level of WNT-5A expression than LSCC (P = 0.0289, Fisher test), while MMP-3 immunoexpression was statistically more significant in LSCC than in AC (P = 0.0285, Fisher test). Immunolabeling of beta-catenin protein was differentially distributed between samples; the majority of AC cases (61.90%) demonstrated a membranous-cytoplasmic pattern, while a considerable number of LSCC cases (29.41%) revealed a cytoplasmic pattern, instead of the usual membranous pattern. Conclusions: The present results suggest that WNT-5A may be an important marker during initial events of AC malignant transformation, in which non-canonical and canonical Wnt/beta-catenin signaling pathways could be involved. Additionally, WNT-5A might recruit other events in LSCC, such as MMP-3 protein synthesis, as its presence is increased in established malignant processes without beta-catenin dependency.

Peroxiredoxin I is differentially expressed in multiple myelomas and in plasmablastic lymphomas

BACKGROUND: Plasmablastic lymphoma (PBL) and multiple myeloma (MM) are B cell-derived malignancies that share many morphologic and immunophenotypic traits, making the differential diagnosis particularly complicated. We have recently demonstrated that peroxiredoxin I (PrdxI) is expressed in plasma cells but not in B lymphocytes, suggesting that its expression is development-associated. AIM: To analyze PrdxI expression in PBL and in MM in order to study its utilization as an additional diagnostic molecular tool. METHODS AND RESULTS: Eight cases of PBL and nine of MM were studied by immunohistochemistry. We have demonstrated that PrdxI expression is closely connected with the immunoglobulin production capacity of the cells, which means high in MM, but absent in PBL cases, except one, wherein few cells were stained. CONCLUSIONS: We hypothesize PrdxI as a component of the unfolded protein response (UPR), an adaptive pathway essential for plasma cell differentiation. As we have not detected immunoglobulin in our PBL cases, we suggest that UPR was not activated in the cells, accounting for the impediment of the developmental process, and for the inhibition of PrdxI expression observed. PrdxI could be considered an additional plasma cell functional marker and could also be speculated as a therapeutic target in the treatment of MM. Oral Diseases (2008) 14, 741-746

Correlation between c-Jun and human papillomavirus in oral premalignant and malignant lesions

c-Jun, one of the components of the transcription factor activating protein-1 (AP-1), is suggested as a factor in malignant progression of oral lesions. c-Jun and other AP-1 components relationships with human papillomavirus (HPV) infection have been investigated, but not yet focusing on oral carcinogenesis. The aim of this study was to verify whether c-Jun immunohistochemical expression is related to HPV DNA detection in oral premalignant and malignant lesions. Fifty cases diagnosed as oral leukoplakias, with different degrees of epithelial dysplasia, and as oral squamous cell carcinomas (OSCC) were submitted to immunohistochemistry to detect c-Jun and to in situ hybridization with signal amplification to assess HPV DNA. It was verified that c-Jun nuclear expression increased according to the degree of dysplasia within the lesion, with the greatest expression in OSCC. The same did not happen concerning HPV infection - a discrete proportional relation was observed in indexes found in leukoplakia with no dysplasia, leukoplakia with dysplasia and OSCC, but statistically insignificant. When separating the group of leukoplakia by degrees of dysplasia, this relation of proportion was not observed. Nevertheless, the overall prevalence of HPV infection was 24% and the high-risk HPV types were the most frequently identified, which does not allow excluding HPV as a risk factor in oral carcinogenesis. When relating c-Jun expression and HPV infection, no statistically significant relationship is observed. Results suggest then that malignant progression mediated by c-Jun is independent of the presence of HPV in oral carcinogenesis. (C) 2007 Elsevier Ltd. All rights reserved.

Response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate membrane

This study investigated the response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane. Osteoblastic cells were cultured in osteogenic conditions either on P(VDF-TrFE)/BT or polytetrafluoroethylene (PTFE) for up to 14 days. At 7 and 14 days, the mRNA expression of Runt-related transcription factor 2 (RUNX2), Type I collagen (COL I), Osteopontin (OPN), Alkaline phosphatase (ALP), Bone sialoprotein (BSP), and Osteocalcin (OC), key markers of the osteoblastic phenotype, and of Bcl2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and Survivin (SUR), associated with the control of the apoptotic cell death, was assayed by real-time PCR. In situ ALP activity was qualitatively evaluated by means of Fast red staining. Surface characterization was also qualitatively and quantitatively assayed in terms of topography, roughness, and wettability. Cells grown on P(VDF-TrFE)/BT exhibited a significantly higher mRNA expression for all markers compared to the ones on PTFE, except for Bcl-2, which was not detected for both groups. Additionally, Fast red staining was noticeably stronger in cultures on P(VDF-TrFE)/BT at 7 and 14 days. At micron-and submicron scale, SEM images and roughness analysis revealed that PTFE and P(VDF-TrFE)/BT exhibited a smooth topography and a similar roughness, respectively. PTFE membrane displayed higher contact angles compared with P(VDF-TrFE)/BT, as indicated by wettability assay. The novel P(VDF-TrFE)/BT membrane supports the acquisition of the osteoblastic phenotype in vitro, while up-regulating the expression of apoptotic markers. Further in vivo experiments should be carried out to confirm the capacity of P(VDF-TrFE)/BT membrane in promoting bone formation in guided bone regeneration.