Stephano S. Mello

Gene expression profiles in human lymphocytes irradiated in vitro with low doses of gamma rays

Abstract Fachin, A. L., Mello, S. S., Sandrin-Garcia, P., Junta, C. M., Donadi, E. A., Passos, G. A. S. and Sakamoto-Hojo, E. T. Gene Expression Profiles in Human Lymphocytes Irradiated In Vitro with Low Doses of Gamma Rays. Radiat. Res. 168, 650–665 (2007). The molecular mechanisms underlying responses to low radiation doses are still unknown, especially in normal lymphocytes, despite the evidence suggesting specific changes that may characterize cellular responses. Our purpose was to analyze gene expression profiles by DNA microarrays in human lymphocytes after in vitro irradiation (10, 25 and 50 cGy) with γ rays. A cytogenetic analysis was also carried out for different radiation doses. G0 lymphocytes were irradiated and induced to proliferate for 48 h; then RNA samples were collected for gene expression analysis. ANOVA was applied to data obtained in four experiments with four healthy donors, followed by SAM analysis and hierarchical clustering. For 10, 25 and 50 cGy, the numbers of significantly (FDR ≤ 0.05) modulated genes were 86, 130 and 142, respectively, and 25, 35 and 33 genes were exclusively modulated for each dose, respectively. We found CYP4X1, MAPK10 and ATF6 (10 cGy), DUSP16 and RAD51L1 (25 cGy), and RAD50, REV3L and DCLRE1A (50 cGy). A set of 34 significant genes was common for all doses; while SERPINB2 and C14orf104 were up-regulated, CREB3L2, DDX49, STK25 and XAB2 were down-regulated. Chromosome damage was significantly induced for doses ≥10 cGy (total aberrations) and ≥50 cGy (dicentrics/ rings). Therefore, low to moderate radiation doses induced qualitative and/or quantitative differences and similarities in transcript profiles, reflecting the type and extent of DNA lesions. The main biological processes associated with modulated genes were metabolism, stress response/DNA repair, cell growth/differentiation, and transcription regulation. The results indicate a potential risk to humans regarding the development of genetic instability and acquired diseases.

Immunosuppressive therapy modulates T lymphocyte gene expression in patients with systemic lupus erythematosus

To evaluate the T‐cell large‐scale differential gene expression in systemic lupus erythematosus (SLE) patients presenting with glomerulonephritis we studied SLE patients before and after immunosuppressive treatment. Large‐scale gene expression of peripheral blood mononuclear T cells was evaluated using cDNA microarray nylon membranes containing 5184 cDNAs. Data were analysed using the SAM and Cluster and Treeview software. When untreated patients were compared to healthy individuals, 38 genes, most of them located on chromosomes 1, 3, 6, 17 and 19, were repressed, and when untreated patients were compared to treated ones, 154 genes, located on chromosomes 1, 6, 7, 12 and 17, were induced. In terms of biological function of coded proteins, the differentially expressed genes were associated with apoptosis, cell cycle, chromosomal scaffold, cytokine/chemokine, DNA repair/replication, Golgi/mitochondrial proteins, mRNA processing, signalling molecules and tumour suppressors. Two autoantigen genes related to RNA splicing (small nuclear riboprotein 70 000 MW‐U1 SNR, and splicing factor 3a, 60 000 MW), and the tetranectin‐plasminogen‐binding protein were repressed. The Fc fragment of immunoglobulin G low affinity IIb, apoptotic protease activating factor‐1, two subunits of cytochrome c, caspase 8, complement C5a, HLA‐DRA, HLA‐DQB1, transforming growth factor‐β receptor II, small nuclear ribonucleoprotein polypeptide N (Sm protein N) genes, heterogeneous nuclear riboprotein‐C, and argininosuccinate lyase genes, among others, were induced. A total of 10 genes were repressed in untreated patients and induced in treated ones, among them tumour necrosis factor (ligand) superfamily member 9, tumour protein p53, mannosidase alpha class IA, and CD22. Although some of these differentially expressed genes are typically expressed in B cells, CD22 and CD32 have also been reported in T cells and may provide regulatory signals to B cells. Assessment of differential gene expression may provide hybridization signatures that may identify susceptibility, diagnostic and prognostic markers of SLE.

Differential gene expression of peripheral blood mononuclear cells from rheumatoid arthritis patients may discriminate immunogenetic, pathogenic and treatment features

This study aimed to evaluate the association between the differential gene expression profiling of peripheral blood mononuclear cells of rheumatoid arthritis patients with their immunogenetic (human leucocyte antigen shared‐epitope, HLA‐SE), autoimmune response [anti‐cyclic citrullinated peptide (CCP) antibodies], disease activity score (DAS‐28) and treatment (disease‐modifying antirheumatic drugs and tumour necrosis factor blocker) features. Total RNA samples were copied into Cy3‐labelled complementary DNA probes, hybridized onto a glass slide microarray containing 4500 human IMAGE complementary DNA target sequences. The Cy3‐monocolour microarray images from patients were quantified and normalized. Analysis of the data using the significance analysis of microarrays algorithm together with a Venn diagram allowed the identification of shared and of exclusively modulated genes, according to patient features. Thirteen genes were exclusively associated with the presence of HLA‐SE alleles, whose major biological function was related to signal transduction, phosphorylation and apoptosis. Ninety‐one genes were associated with disease activity, being involved in signal transduction, apoptosis, response to stress and DNA damage. One hundred and one genes were associated with the presence of anti‐CCP antibodies, being involved in signal transduction, cell proliferation and apoptosis. Twenty‐eight genes were associated with tumour necrosis factor blocker treatment, being involved in intracellular signalling cascade, phosphorylation and protein transport. Some of these genes had been previously associated with rheumatoid arthritis pathogenesis, whereas others were unveiled for future research.

Comprehensive gene expression profiling in lungs of mice infected with Mycobacterium tuberculosis following DNAhsp65 immunotherapy

The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR‐TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood.

The non-coding RNA BC1 is down-regulated in the hippocampus of Wistar Audiogenic Rat (WAR) strain after audiogenic kindling

The aim of this study was to identify molecular pathways involved in audiogenic seizures in the epilepsy-prone Wistar Audiogenic Rat (WAR). For this, we used a suppression-subtractive hybridization (SSH) library from the hippocampus of WARs coupled to microarray comparative gene expression analysis, followed by Northern blot validation of individual genes. We discovered that the levels of the non-protein coding (npc) RNA BC1 were significantly reduced in the hippocampus of WARs submitted to repeated audiogenic seizures (audiogenic kindling) when compared to Wistar resistant rats and to both naive WARs and Wistars. By quantitative in situ hybridization, we verified lower levels of BC1 RNA in the GD-hilus and significant signal ratio reduction in the stratum radiatum and stratum pyramidale of hippocampal CA3 subfield of audiogenic kindled animals. Functional results recently obtained in a BC1⁻/⁻ mouse model and our current data are supportive of a potential disruption in signaling pathways, upstream of BC1, associated with the seizure susceptibility of WARs.

Shared and unique gene expression in systemic lupus erythematosus depending on disease activity.

Patients presenting with active systemic lupus erythematosus (SLE) manifestations may exhibit distinct pathogenetic features in relation to inactive SLE. Also, cDNA microarrays may potentially discriminate the gene expression profile of a disease or disease variant. Therefore, we evaluated the expression profile of 4500 genes in peripheral blood lymphocytes (PBL) of SLE patients. We studied 11 patients with SLE (seven with active SLE and four with inactive SLE) and eight healthy controls. Total RNA was isolated from PBL, reverse transcribed into cDNA, and postlabeled with Cy3 fluorochrome. These probes were then hybridized to a glass slide cDNA microarray containing 4500 human IMAGE cDNA target sequences. An equimolar amount of total RNA from human cell lines served as reference. The microarray images were quantified, normalized, and analyzed using the R environment (ANOVA, significant analysis of microarrays, and cluster‐tree view algorithms). Disease activity was assessed by the SLE disease activity index. Compared to the healthy controls, 104 genes in active SLE patients (80 repressed and 24 induced) and 52 genes in nonactive SLE patients (31 induced and 21 repressed) were differentially expressed. The modulation of 12 genes, either induced or repressed, was found in both disease variants; however, each disease variant had differential expression of different genes. Taken together, these results indicate that the two lupus variants studied have common and unique differentially expressed genes. Although the biological significance of the differentially expressed genes discussed above has not been completely understood, they may serve as a platform to further explore the molecular basis of immune deregulation in SLE.

Delayed effects of exposure to a moderate radiation dose on transcription profiles in human primary fibroblasts

Ionizing radiation (IR) is used in a wide variety of medical and nonmedical applications and poses a potential threat to human health. Knowledge of changes in gene expression in irradiated cells may be helpful for the establishment of effective paradigms for radiation protection. IR‐induced DNA damage triggers a complex cascade of signal transduction. Recently, genome‐wide approaches have allowed the detection of alterations in gene expression across a wide range of radiation doses. However, the delayed or long‐term biological effects of mild‐doses of IR remain largely unknown. The main objective of the present study was to investigate the effects of a moderate dose of gamma‐rays (50 cGy) on gene expression 6 days post‐irradiation. Gene expression using cDNA microarrays revealed statistically significant changes in the expression of 59 genes (FDR < 0.07), whose functions are related to cell‐cycle control, protein trafficking, ubiquitin cycle, Rho‐GTPAse pathway, protein phosphatase signalization, oxidoreductase control, and stress response. A set of 464 genes was also selected by a less stringent approach, and we demonstrate that this broader set of genes can efficiently distinguish the irradiated samples from the unirradiated, defining a long‐term IR signature in human primary fibroblasts. Our findings support the existence of persistent responses to mild doses of IR detectable by changes in gene expression profiles. These results provide insight into delayed effects observed in human primary cells as well as the role of long‐term response in neoplastic transformation. Environ. Mol. Mutagen. 52:117–129, 2011.

Gene Expression Profiles Stratified according to Type 1 Diabetes Mellitus Susceptibility Regions

The MHC region (6p21) aggregates the major genes that contribute to susceptibility to type 1 diabetes (T1D). Three additional relevant susceptibility regions mapped on chromosomes 1p13 (PTPN22), 2q33 (CTLA‐4), and 11p15 (insulin) have also been described by linkage studies. To evaluate the contribution of these susceptibility regions and the chromosomes that house these regions, we performed a large‐scale differential gene expression on lymphomononuclear cells of recently diagnosed T1D patients, pinpointing relevant modulated genes clustered in these regions and their respective chromosomes. A total of 4608 cDNAs from the IMAGE library were spotted onto glass slides using robotic technology. Statistical analysis was carried out using the SAM program, and data regarding gene location and biological function were obtained at the SOURCE, NCBI, and FATIGO programs. Three induced genes were observed spanning around the MHC region (6p21–6p23), and seven modulated genes (5 repressed and 2 repressed) were seen spanning around the 6q21–24 region. Additional modulated genes were observed in and around the 1p13, 2q33, and 11p15 regions. Overall, modulated genes in these regions were primarily associated with cellular metabolism, transcription factors and signaling transduction. The differential gene expression characterization may identify new genes potentially involved with diabetes pathogenesis.

Alterations in gene expression profiles correlated with cisplatin cytotoxicity in the glioma U343 cell line

Gliomas are the most common tumors in the central nervous system, the average survival time of patients with glioblastoma multiforme being about 1 year from diagnosis, in spite of harsh therapy. Aiming to study the transcriptional profiles displayed by glioma cells undergoing cisplatin treatment, gene expression analysis was performed by the cDNA microarray method. Cell survival and apoptosis induction following treatment were also evaluated. Drug concentrations of 12.5 to 300 μM caused a pronounced reduction in cell survival rates five days after treatment, whereas concentrations higher than 25 μM were effective in reducing the survival rates to ~1%. However, the maximum apoptosis frequency was 20.4% for 25 μM cisplatin in cells analyzed at 72 h, indicating that apoptosis is not the only kind of cell death induced by cisplatin. An analysis of gene expression revealed 67 significantly (FDR < 0.05) modulated genes: 29 of which down- and 38 up-regulated. These genes belong to several classes (metabolism, protein localization, cell proliferation, apoptosis, adhesion, stress response, cell cycle and DNA repair) that may represent several affected cell processes under the influence of cisplatin treatment. The expression pattern of three genes (RHOA, LIMK2 and TIMP2) was confirmed by the real time PCR method.

Portrait of Transcriptional Expression Profiles Displayed by Different Glioblastoma Cell Lines

Glioblastoma multiforme (GBM) is among the most lethal of all human tumors, with the average survival of approximately 1 year from diagnosis (Avgeropoulos and Batchelor, 1999). In glioblastomas, LOH 10q is the most frequent genetic alteration (69%), followed by EGFR amplification (34%), TP53 mutations (31%), p16INK4a deletion (31%), and PTEN mutations (24%)(Ohgaki and Kleihues, 2005). Treatments of patients with GBM include surgery, radiotherapy and parallel adjuvant chemotherapy (Stupp et al., 2005). While radiotherapy has been found to significantly prolong survival rates for GBM patients, a poor prognosis and radioresistance are typical characteristics of this disease (Stupp et al., 2007). The antitumoral drug Temozolomide (TMZ) constitutes, in combination with radiotherapy, the current standard of care for glioblastoma (Stupp et al., 2005). However, the action of TMZ may be counteracted in tumors by the expression of the DNA repair enzyme MGMT, which repairs TMZ-induced DNA lesions (Hegi et al., 2005). MGMT activity and resistance to TMZ were highly correlated, indicating that MGMT is a major predictor of response to TMZ in glioma cells (Hermisson et al., 2006). According to the authors, collectively, MGMT expression and TP53 status may become valuable parameters to predict cell responses to TMZ treatment in patients with GBM. Modulation of MSH6, PARP1 and NTL1, DNA repair genes involved in mismatch repair and base excision repair (BER), were found at transcription and protein levels in GBM cells resistant to TMZ (Zhang et al., 2010). Other genotoxic agents exert different effects in GBM cells with different TP53 status. UV light (UV-C) (Batista et al., 2009) and chloroethylating agents (ACNU and BCNU) (Batista et al., 2007), significantly induce apoptosis in TP53-mutated glioma cells, while WT TP53 cells are more sensitive to methylating agents, including TMZ (Hermisson et al., 2006; Roos et al., 2007). Efforts have been made to overcome drug and radio-resistance of GBM cells, but the heterogeneity of these tumors seems to be critical, and molecular analysis is an important tool to elucidate the mechanisms underlying cellular responses to antitumoral agents. Recently, application of genome-scale methodologies has opened the opportunity to study