ognizant Communication Corporation

GENE EXPRESSION

ABSTRACTS
VOLUME 13, NUMBER 1

Gene Expression, Vol. 13, pp. 1-14
1052-2166/06 $20.00 + .00
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Vascular Endothelial Growth Factor (VEGF) Is Suppressed in WT1-Transfected LNCaP Cells

Kylie Graham,1 Wenliang Li,2 Bryan R. G. Williams,2 and Gail Fraizer1

1Department of Biological Sciences, Kent State University, Kent, OH 44242
2Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195

The Wilms' tumor suppressor gene product (WT1) regulates expression of growth control genes. Microarray analysis of gene expression profiles of hormone-treated LNCaP prostate cancer cell lines transfected with either wild-type WT1 or a zinc finger mutant form, DDS (R394W), revealed significantly altered patterns of expression. Validation studies using quantitative real-time PCR confirmed the differential expression of the tumor progression gene, vascular endothelial growth factor (VEGF). WT1-LNCaP cells had significantly reduced levels of VEGF mRNA when compared to vector control cells; in contrast, DDS-LNCaP cells showed elevated levels of VEGF transcripts. To address a functional role for WT1 overexpression, we investigated whether induction of VEGF expression, by the synthetic androgen R1881, would be disrupted in wild-type or mutant WT1-transfected LNCaP cells. Hormone treatment failed to elevate VEGF transcript levels above uninduced baseline levels in WT1-LNCaP cells, despite 48 h of treatment with 5 nM R1881. Consistent with our quantitative real-time PCR analysis, immunofluorescent staining of VEGF protein was reduced in WT1-LNCaP cells in both the presence and absence of R1881 treatment. Conversely, VEGF levels increased in vector control and DDS-LNCaP cells treated with 5 nM R1881. Not only do these studies point out the regulatory potential of WT1 for VEGF, but they also indicate an altered function for the mutant DDS isoform. Because VEGF is associated with neovascularization and promotion of metastasis in a variety of solid tumors including prostate cancer, a better understanding of the regulation of VEGF expression by transcription factors, such as WT1, is important for halting disease progression.

Key words: Prostate cancer; Wilms' tumor gene; Vascular endothelial growth factor; Denys-Drash syndrome; Microarray

Address correspondence to Gail Fraizer, Department of Biological Sciences, 251 Cunningham Hall, Kent State University, Kent, OH 44242, USA. Tel: 330-672-1398; Fax: 330-672-3713; E-mail: gfraizer@kent.edu




Gene Expression, Vol. 13, pp. 15-26
1052-2166/06 $20.00 + .00
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Gene Expression Profiles of the Aurora Family Kinases

Yong-Shiang Lin,1* Li-Jen Su,1,2* Chang-Tze Ricky Yu,1* Fen-Hwa Wong,4* Hsu-Hua Yeh,1 Su-Liang Chen,1 Jiunn-Chyi Wu,1 Wey-Jinq Lin,5 Yow-Ling Shiue,6 Hsiao-Sheng Liu,7 Shih-Lan Hsu,8 Jin-Mei Lai,9 and Chi-Ying F. Huang1,2,3,10

1Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli County 350, Taiwan, R.O.C.
2Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan, R.O.C.
3Institute of Biotechnology in Medicine, National Yang-Ming University, Taipei 112, Taiwan, R.O.C.
4Institute of Public Health, National Yang-Ming University, Taipei 112, Taiwan, R.O.C.
5Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei 112, Taiwan, R.O.C.
6Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung 804, Taiwan, R.O.C.
7Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan, R.O.C.
8Department of Education and Research, Taichung Veterans General Hospital, Taichung 400, Taiwan, R.O.C.
9Department of Life Science, Fu-Jen Catholic University, Taipei Hsien 242, Taiwan, R.O.C.
10Department of Computer Science and Information Engineering, National Taiwan University, Taipei 106, Taiwan, R.O.C.

The evolutionarily conserved Aurora family kinases, a family of mitotic serine/threonine kinases, has three members in humans (Aurora-A, -B and -C). Overexpression of Aurora family members, particularly Aurora-A, has been reported in many human cancers and cell lines. In this study, we present evidence based on comparative gene expression analysis via quantitative RT-PCR to delineate the relative contributions of these kinases in 60 cell lines and statistical analysis in five different human cancer microarray datasets. The analysis demonstrated the selective upregulation of these Aurora members in various cancers. In general, Aurora-A exhibited the highest expression levels, with substantially decreased quantities of the Aurora-C transcript detected relative to Aurora-A and-B. Moreover, to characterize the roles of each Aurora member, which share many similarities, we investigated the expression profiles of the family in normal tissues and a panel of different phases of the HeLa cell cycle. Finally, both Aurora-A and -B were overexpressed in a majority of esophageal tumor tissues in comparison to the normal variants. Taken together, the results show that each Aurora member exhibits distinct expression patterns, implying that they are engaged in different biological processes to accomplish more elaborate cell physiological functions in higher organisms.

Key words: Aurora family; Aurora-A; Aurora-B; Aurora-C; Esophageal carcinoma; Quantitative RT-PCR

Address correspondence to Chi-Ying F. Huang, Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli County 350, Taiwan, R.O.C. Tel: 886-37-246-166, ext. 35305; Fax: 886-37-586-459; E-mail: chiying@nhri.org.tw

*These authors contributed equally.




Gene Expression, Vol. 13, pp. 27-40
1052-2166/06 $20.00 + .00
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Molecular Cloning of the m-Golsyn Gene and its Expression in the Mouse Brain

Eishi Funakoshi,1 Ayako Hamano,1 Masaki Fukui,1 Norito Nishiyama,2 Kiyokazu Ogita,2 Nobuyoshi Shimizu,3 and Fumiaki Ito1

1Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan
2Department of Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan
3Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

The mouse ortholog of the human GOLSYN gene, termed the m-Golsyn gene, was isolated and mapped to the region on mouse chromosome 15B3.2 syntenic with human chromosome 8q23. Three mRNA species (type 1a, 1b, and type 2) were produced by use of alternative transcription initiation points and alternative splicing events. The type 1 mRNAs were expressed only in the brain, whereas the type 2 was detected in various tissues. m-Golsyn protein was expressed in various tissues including the brain. Immunohistochemical study of m-Golsyn protein showed its prominent expression in the neuronal cells in various regions of the brain and strong expression in the choroid plexus ependymal cells lining the ventricles. m-Golsyn protein was found to be homologous to syntaphilin, a regulator of synaptic vesicle exocytosis. These results indicate that the m-Golsyn protein may play an important role in intracellular protein transport in neuronal cells of the brain.

Key words: GOLSYN; m-Golsyn; Brain; Protein trafficking; Syntaphilin; Neuronal cells; Genomic structure; Choroid plexus; Chromosome 15B3.2; cDNA cloning

Address correspondence to Eishi Funakoshi, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka 573-0101, Japan. Tel: +81-72-866-3118; Fax: +81-72-866-3117; E-mail: funakos@pharm.setsunan.ac.jp




Gene Expression, Vol. 13, pp. 41-52
1052-2166/06 $20.00 + .00
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Elucidation of Stannin Function Using Microarray Analysis: Implications for Cell Cycle Control

Brian E. Reese,1 Dan Krissinger,1 Jong K. Yun,1,2 and Melvin L. Billingsley1

1Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA
2Jake Gittlen Cancer Research Institute, Penn State College of Medicine, Hershey, PA 17033, USA

Stannin (Snn) is a highly conserved, vertebrate protein whose cellular function is unclear. We have recently demonstrated in human umbilical vein endothelial cells (HUVECs) that Snn gene expression is significantly induced by tumor necrosis factor-a (TNF-a) in a protein kinase C-e (PKC-e)-dependent manner. In HUVEC, TNF-a stimulation of HUVECs results in altered gene expression, and a slowing or halting of cell growth. An initial set of experiments established that Snn knockdown via siRNA, prior to TNF-a treatment, resulted in a significant inhibition of HUVEC growth compared to TNF-a treatment alone. In order to assess how Snn may be involved in TNF-a signaling in HUVEC growth arrest, we performed microarray analysis of TNF-a-stimulated HUVECs with and without Snn knockdown via siRNA. The primary comparison made was between TNF-a-stimulated HUVECs and TNF-a-exposed HUVECs that had Snn knocked down via Snn-specific siRNAs. Ninety-six genes were differentially expressed between these two conditions. Of particular interest was the significant upregulation of several genes associated with control of cell growth and/or the cell cycle, including interleukin-4, p29, WT1/PRKC, HRas-like suppressor, and MDM4. These genes act upon cyclin D1 and/or p53, both of which are key regulators of the G1 phase of the cell cycle. Functional studies further supported the role of Snn in cell growth, as cell cycle analysis using flow cytometry shows a significant increase of G1 cell cycle arrest in HUVECs with Snn knockdown in response to TNF-a treatment. Together these studies suggest a functional role of Snn in regulation of TNF-a-induced signaling associated with HUVEC growth arrest.

Key words: Stannin; TNF-a; HUVEC; Microarray; Cell growth; Cell cycle

Address correspondence to Mel Billingsley, Department of Pharmacology, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA. Tel: 717-635-2100; Fax: 717-531-5013; E-mail: mlb8@psu.edu




Gene Expression, Vol. 13, pp. 53-57
1052-2166/06 $20.00 + .00
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Ontogenetic Distribution of 5-HT2C, 5-HT5A, and 5-HT7 Receptors in the Rat Hippocampus

Guadalupe García-Alcocer,1 Laura Cristina Berumen Segura,1 Mariane García Peña,1 Ataúlfo Martínez-Torres,2 and Ricardo Miledi2,3

1Facultad de Química, Universidad Autónoma de Querétaro, Centro Universitario, Querétaro 76010, México
2Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Apartado Postal 1-1141, Juriquilla Querétaro 76001, México
3Department of Neurobiology and Behavior, U. C. Irvine, CA 92697, USA

It is known that serotonin exerts its different nociceptive and motor functions by interacting with distinct receptors subtypes, which could be either G-protein coupled or ionotropic. Previous reports demonstrated the early activation of serotonin receptor transcripts during rat development, suggesting a potential role of the serotoninergic system during ontogeny. In this study we have compared the cellular distribution of three serotonin receptor subtypes: 5-HT2C, 5-HT5A, and 5-HT7. Immunocytochemical methods were used in slices of rat hippocampus obtained during the postnatal development. 5-HT2C immunoreactivity was strong at all developmental stages in the CA1 region, whereas differences were observed between P0 and P5 in the CA3 region. The 5-HT5A receptor immunosignal in CA1 and CA3 was strong at P0, decreased at P11, and then increased in the adult. The immunoreactivity to 5-HT7 receptors was high in all regions at P0 and then decreased progressively during postnatal development; the signal was stronger for 5-HT2C than for 5-HT5A and 5-HT7 receptors. Changes in the expression level of each receptor may result in differences in functional and pharmacological properties of the cells expressing them as well as in the hippocampal neuronal network. The distribution of the three serotonin receptor subtypes studied varied during the ontogeny, which supports their potential role during development and will help to understand their mechanisms.

Key words: Serotonin receptors; 5-HT5A receptors; 5-HT7 receptors; Hippocampus development

Address correspondence to Guadalupe García-Alcocer, Facultad de Química, Universidad Autónoma de Querétaro, Cerro de las Campanas, C.P. 76010, Querétaro, México. Tel: 52 44 21 92 12 67; Fax: 52 44 21 92 13 02; E-mail: leguga@email.com




Gene Expression, Vol. 13, pp. 59-
1052-2166/06 $20.00 + .00
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Rare Functional Variants of Podocin (NPHS2) Promoter in Patients With Nephrotic Syndrome

Roberta Oleggini,1* Roberta Bertelli,1* Armando Di Donato,1 Marco Di Duca,1 Gianluca Caridi,1 Simone Sanna-Cherchi,2 Francesco Scolari,3 Luisa Murer,4 Landino Allegri,2 Rosanna Coppo,5 Francesco Emma,6 Giovanni Camussi,7 Francesco Perfumo,8 and Gian Marco Ghiggeri1

1Laboratory on Pathophysiology of Uremia, Istituto G. Gaslini, Genoa, Italy
2Department of Clinical Medicine, Nephrology and Health Sciences, University of Parma, Italy
3Unit of Nephrology, Spedali Civili di Brescia, Italy
4Department of Pediatrics, University of Padua, Italy
5Nephrology Section, Ospedale Regina Margherita, Turin, Italy
6Nephrology Section, Ospedale Bambin Gesù, Rome, Italy
7Chair of Nephrology, University of Turin, Italy
8Section of Nephrology, Istituto G. Gaslini, Genoa, Italy

Podocin (NPHS2) is a component of the glomerular slit-diaphragm, with major regulatory functions in renal permeability of proteins. Loss of podocin and decrease in resynthesis may influence the outcome of proteinuric renal disease such as segmental glomerulosclerosis (FSGS), and promoter functionality plays a key role in this process. NPHS2 promoter variants with functional activity may be a part of the problem of podocin resynthesis. We sequenced NPHS2 promoter region from -628 to ATG in a large cohort of 260 nephrotic patients (161 with FSGS) who were presenting proteinuria from moderate to severe and were receiving or had received modular therapies according to their sensitivity to steroids and other immune modulators. Three sequence variants (-236C>T, -52C>G, -26C>G) were identified in our study population that gave an allele frequency below 1% (5 patients out of 520 alleles). Functional implications were shown for each variants that were most evident for -52C>G and -26C>G (-50% of luciferase expression compared to the wild-type sequence, p < 0.01). Consensus analysis for homology of the -52 region with regulatory factors revealed homology for USF1 and the sum of experiments with gel retardation and with cells silenced for USF1 confirmed that this factor regulates NPHS2 expression at this site. In conclusion, three functional variants in NPHS2 promoter have been identified in a large cohort of patients with nephrotic syndrome and FSGS that have a frequency <1%. One of these (i.e., -52C>G) is associated with a poor clinical outcome and evolution to end-stage renal failure. USF1 was identified as the transcriptional factor regulating NPHS2 at this site. Even if not sufficient to cause FSGS per se, these variants could represent modifiers for severity and/or progression of the disease.

Key words: NPHS2 gene; NPHS2 promoter; Nephrotic syndrome; Focal segmental glomerulosclerosis; Proteinuria; Podocin; USF1

Address correspondence to Gian Marco Ghiggeri, M.D., Laboratorio di Fisiopatologia dell'Uremia, Istituto G.Gaslini, 16148 Genova, Italy. Tel: ++39 010 380742; Fax: ++39 010 395214; E-mail: labnefro@ospedale-gaslini.ge.it

*Roberta Oleggini and Roberta Bertelli contributed equally to the study.