Biomedical subjects

Anthony Galante

Publications and source records attributed to Anthony Galante.

4 recordsLinked to original sources

PubMed2006

Involvement of HTLV-I Tax and CREB in aneuploidy: a bioinformatics approach.

BACKGROUND: Adult T-cell leukemia (ATL) is a complex and multifaceted disease associated with human T-cell leukemia virus type 1 (HTLV-I) infection. Tax, the viral oncoprotein, is considered a major contributor to cell cycle deregulation in HTLV-I transformed cells by either directly disrupting cellular factors (protein-protein interactions) or altering their transcription profile. Tax transactivates these cellular promoters by interacting with transcription factors such as CREB/ATF, NF-kappaB, and SRF. Therefore by examining which factors upregulate a particular set of promoters we may begin to understand how Tax orchestrates leukemia development. RESULTS: We observed that CTLL cells stably expressing wild-type Tax (CTLL/WT) exhibited aneuploidy as compared to a Tax clone deficient for CREB transactivation (CTLL/703). To better understand the contribution of Tax transactivation through the CREB/ATF pathway to the aneuploid phenotype, we performed microarray analysis comparing CTLL/WT to CTLL/703 cells. Promoter analysis of altered genes revealed that a subset of these genes contain CREB/ATF consensus sequences. While these genes had diverse functions, smaller subsets of genes were found to be involved in G2/M phase regulation, in particular kinetochore assembly. Furthermore, we confirmed the presence of CREB, Tax and RNA Polymerase II at the p97Vcp and Sgt1 promoters in vivo through chromatin immunoprecipitation in CTLL/WT cells. CONCLUSION: These results indicate that the development of aneuploidy in Tax-expressing cells may occur in response to an alteration in the transcription profile, in addition to direct protein interactions.

Aneuploidy↗

PubMed2005

Global gene profiling reveals novel glucocorticoid induced changes in gene expression of human lens epithelial cells.

PURPOSE: Prolonged use of glucocorticoids can lead to the formation of a cataract, however the mechanism is not known. We recently reported the presence of the functional glucocorticoid receptor in immortalized cultured mammalian lens epithelial cells (LECs), but the biological effect is not known. This study seeks to determine if freshly isolated human LECs respond to glucocorticoid treatment and to examine glucocorticoid induced changes in global gene expression in LECs. METHODS: Capsulorhexis specimens obtained in surgery from eyes with cataract were cultured. Primary lens cultures were transfected, in triplicate, with pGRE.Luc, which drives the expression of firefly luciferase, and treated with dexamethasone (Dex) or vehicle (Veh). RNA isolated from HLE B-3 cells, treated with Dex or Veh for 4 or 16 h in triplicate, was used to analyze global changes in gene expression by microarray hybridization. Data and cluster analyses were performed using Microarray Suite 5.0, GeneSpring 6.1, EASE, NetAffx, and SAM. Real Time PCR was used to confirm microarray data in RNA isolated from HLE B-3 cells in triplicate and a primary culture of human lens epithelial cells. RESULTS: Transfected primary cultures of human LECs treated with Dex demonstrated a glucocorticoid response with a greater than 4 fold increase in firefly luciferase activity over controls. Microarray data revealed that 136 genes were modulated with 4 h treatment with Dex. Of the 136 genes, 93 transcripts were upregulated and 43 were downregulated by greater than 1.5 fold. Eighty-six genes were modulated with 16 h Dex treatment. Of the 86 genes, 30 transcripts were upregulated and 56 were downregulated by greater than 1.5 fold. Microarray results were verified by Real Time PCR in both the HLE B-3 and primary cultures of lens epithelial cell. CONCLUSIONS: The activation of a GRE reporter gene in primary cultures of human LECs demonstrates that the glucocorticoid receptor is functional in non-immortalized human lens cells. Microarray studies at 2 time periods demonstrate that glucocorticoids modulate gene expression in immortalized human LECs, reveal novel changes in gene expression, and confirm an endogenous genomic lens glucocorticoid response. This study demonstrates that primary cultures of lens epithelial cells and microarray technology can be used to determine pathways involved in a lens glucocorticoid response and lead to a better understanding of the formation of a steroid induced cataract.

Cells, Cultured↗

PubMed2005

Using DNA microarray to study human cytomegalovirus gene expression.

DNA microarray technology has become one of the most widely used tools for functional genomics and is playing an ever increasing role in the study of viral infections and host-pathogen interactions. This paper describes the development of an oligonucleotide microarray representing all the predicted open reading frames of the human cytomegalovirus (HCMV) and an established protocol for simultaneously measuring the expression of all HCMV genes. To evaluate the performance of the HCMV array, human foreskin fibroblasts were either mock infected or infected with the HCMV AD169 or Toledo strains. Hybridizations were performed to determine the level of detection of HCMV transcripts from both the AD169 and Toledo strains and to assess reproducibility within and between slides. Overall, approximately 95% of the predicted HCMV genes produced detectable levels of mRNA, with median signal to noise and signal to background ratios of 41 and 14, respectively. Scatter plots of samples within an array and between two arrays resulted in average linear regressions above 0.95 and 0.9, respectively, indicating that data from the arrays are highly reproducible. In addition, transcripts from genes found in the Toledo strain but not in AD169 were specifically detected.

Cell Line↗

PubMed2003

Comprehensive genome-wide comparison of DNA and RNA level scan using microarray technology for identification of candidate cancer-related genes in the HL-60 cell line.

Genome-wide scans for DNA and RNA changes in the HL-60 cell line relative to normal leukocytes were conducted. Microarray-based comparative genome hybridization (CGH) studies were performed with the Spectral Genomics Human Bacterial Artificial Chromosome (BAC) 3MB system. Transcriptional measurements of approximately 12,500 human genes were monitored using Affymetrix U95A GeneChips. In HL-60, genomic DNA amplification of the 8q24 locus, trisomy 18, and deletions at loci 5q11.2 approximately q31, 6q12, 9p21.3 approximately p22, 10p12 approximately p15, 14q22 approximately q31, 17p12 approximately p13.3, and monosomy X were detected. After obtaining locus information about the RNA transcripts from the Affymetrix database, 4368 genes were stratified both according to status of RNA expression and the DNA copy number of their designated loci. The expression level of 2326 (53.25%) of 4368 transcripts is concordant with DNA copy number. Examples of specific, highly expressed, cancer-associated genes in amplified loci include SERPINB10, MYC, TYMS, HEC, and EPB41L3, while CD14, GZMK, TCF7, FOS, MLH3, CTNNA1, IRF1, VIM, CRK, MAP3K1, STAM, MAX, SFRG5, ENC1, PURA, MNT, RASA1, GLRX, UBE2B, NR3C1, PTENP1, BS69, COPEB, SKIP, PIM2, and MIC2 represent cancer-associated genes in deleted loci with decreased expression. The complementary usage of genome-wide DNA and RNA scans should enhance the identification of candidate genes in the neoplastic process.

Adult↗