|ognizant Communication Corporation|
VOLUME 14, NUMBER 4
Gene Expression, Vol. 14, pp. 195-205
1052-2166/08 $90.00 + .00
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Ser484 and Ser494 in REL Are the Major Sites of IKK Phosphorylation In Vitro: Evidence That IKK Does Not Directly Enhance GAL4-REL Transactivation
Michael R. Garbati and Thomas D. Gilmore
Department of Biology, Boston University, Boston, MA, USA
Human c-Rel (REL) is a member of the NF-kB family of transcription factors, and one of its primary physiological roles is in the regulation of B-cell proliferation and survival. Although REL is primarily regulated by cytoplasmic-nuclear translocation through interaction with IkB inhibitors, REL also undergoes several posttranslational modifications that have been proposed to modulate its transcriptional activation activity. For example, phosphorylation of C-terminal sequences of REL has been proposed to increase its transactivation activity. In this report, we have used immune complex kinase assays to identify Ser484 and Ser494 as the primary sites of IKKa- and IKKb-mediated in vitro phosphorylation in the C-terminal transactivation domain of REL. However, in cotransfection studies in A293 cells we have failed to detect IKKb-mediated phosphorylation of these sites on REL in vivo, nor does IKKb appear to interact with REL in these cells. Ser-to-Ala mutation of Ser484 and Ser494 does not affect IKK's ability to enhance GAL4-REL transactivation in reporter gene assays in A293 cells. We also show that the previously reported effects of overexpressed IKK and tumor necrosis factor treatment on GAL4-REL transactivation are due to IKK-mediated activation of the endogenous NF-kB pathway, which increases transcription from kB sites in the promoter of a commonly used GAL4 expression vector. Taken together, these results do not support a role for IKK-mediated phosphorylation as means for regulating the activity of REL in vivo.
Key words: c-Rel; Phosphorylation; IKK; Transactivation; GAL4 reporter assay; NF-kB
Address correspondence to Thomas D. Gilmore, Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA. Tel: 617-353-5444; Fax: 617-353-6340; E-mail: email@example.com
Expression of the p16INK4A/Cdkn2a Gene Is Prevalently Downregulated in Human Pheochromocytoma Tumor Specimens
Peter Muscarella,1,4 Mark Bloomston,1,4 Alexander R. Brewer,1 Anjali Mahajan,2 Wendy L. Frankel,3,4 E. Christopher Ellison,1,4 William B. Farrar,1,4 Christopher M. Weghorst,4,5 and Junan Li4,5
1Department of Surgery, College of Medicine, The Ohio State
University, Columbus, OH, USA
2Department of Chemistry, The Ohio State University, Columbus, OH, USA
3Department of Pathology, College of Medicine, The Ohio State University, Columbus, OH, USA
4Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, OH, USA
5Division of Environmental Health Sciences, College of Public Health, The Ohio State University, Columbus, OH, USA
A number of hereditary syndromes have been found to be associated with pheochromocytoma development, but there is a paucity of data regarding secondary molecular events, such as downregulation of the p16INK4A/Cdkn2a gene (hereafter p16), contributing to pheochromocytoma tumorigenesis. Using tissue microarray and immunohistochemistry, we evaluated the expression of p16 in 31 pheochromocytoma tumor specimens. Our results showed that the p16 gene was expressed at low level or even not expressed in all but one specimens [30/31 (96.8%)], indicative of the prevalence of p16 downregulation in pheochromocytomas. In contrast, high expression of p16 was observed in the majority of control "normal" specimens [5/7 (71.6%)]. To further investigate the molecular mechanisms underlying p16 downregulation in pheochromocytomas, we used quantitative real-time PCR, methylation-specific PCR, and direct DNA sequencing to analyze these specimens for potential genetic alterations of the p16 gene. Deletions and aberrant CpG methylation of p16 were identified in 9 (29.0%) and 11 (35.5%) specimens, respectively, while one specimen harbored a point mutation, Ala -> Pro at residue 20 of P16, and this mutation led to an eightfold decrease in the CDK4-inhibitory activity of P16. The overall frequency of p16 genetic alterations is 67.7%. Taken together, our results demonstrate that reduced expression of p16 is a common event in human pheochromocytomas, and the primary cause for such downregulation is inactivating genetic abnormalities in the p16 gene.
Key words: p16; Pheochromocytomas; Expression downregulation; Genetic alterations
Address correspondence to Dr. Junan Li, Division of Environmental Health Sciences, College of Public Health, The Ohio State University, Columbus, OH 43210, USA. Tel: 614-292-4066; Fax: 614-293-7710; E-mail: firstname.lastname@example.org
Identification of Genes That Exhibit Changes in Expression on the 8p Chromosomal Arm by the Systematic Multiplex RT-PCR (SM RT-PCR) and DNA Microarray Hybridization Methods
Fumiichiro Yamamoto and Miyako Yamamoto
Tumor Development Program, Burnham Institute for Medical Research, La Jolla, CA, USA
Losses of the p-arm of chromosome 8 are frequently observed in breast, prostate, and other types of cancers. Using the Systematic Multiplex RT-PCR (SM RT-PCR) method and the DNA microarray hybridization method, we examined the expression of 273 genes located on the p-arm of chromosome 8 in five breast and three prostate human cancer cell lines. We observed frequent decreases in expression of two dozen genes and increases in expression of several genes on this chromosomal arm. These changes in gene expression of the cell lines were later confirmed by real-time qRT-PCR. Additionally and more importantly, we found that a number of these variations were also observed in the majority of clinical cases of breast cancer we examined. These included downregulation of the MYOM2, NP_859074, NP_001034551, NRG1, PHYIP (PHYHIP), Q7Z2R7, SFRP1, and SOX7 genes, and upregulation of the ESCO2, NP_115712 (GINS4), Q6P464, and TOPK (PBK) genes.
Key words: Systematic Multiplex RT-PCR (SM RT-PCR); Real-time qRT-PCR; DNA microarray hybridization; Chromosome 8p; Chromosomal scanning; Gene expression; Breast cancer
Address correspondence to Fumiichiro Yamamoto, Ph.D., Burnham Institute for Medical Research, 10901 N. Torrey Pines Rd., La Jolla, CA, 92037, USA. Tel: 858-646-3116; Fax: 858-646-3173; E-mail: email@example.com
Temporal Gene Expression Analysis of Human Coronary Artery Endothelial Cells Treated With Simvastatin
Li Qin Zhang,1 Shwu-Fan Ma,2 Dmitry Grigoryev,3 Tera L. Lavoie,2 Hui Qing Xiao,3 Robert Setterquist,4 Hailong Li,1 Jeffrey Jacobson,2 Joe G. N. Garcia,2 and Shui Qing Ye1
1Department of Surgery and Department of Molecular Microbiology
and Immunology, University of Missouri, Columbia, MO, USA
2Department of Medicine, University of Chicago, Chicago, IL, USA
3Department of Medicine, Johns Hopkins University, Baltimore, MD, USA
4Department of Research and Development, Ambion, Inc., Austin, TX, USA
Increasing evidence indicates that the beneficial "pleiotropic" effects of statins on clinical events involve nonlipid mechanisms including the modification of blood vessel endothelial cell function. However, the involved molecular events and pathways are not completely understood. In the present study, Affymetrix microarrays were used to monitor the temporal gene expression of human coronary artery endothelial cells (HCAEC) treated with simvastatin (Sim) to gain insight into statins' direct effects on the endothelial function. We isolated and labeled mRNA from HCAEC treated with Sim for 0, 3, 6, 12, 24, and 48 h and hybridized these samples to Affymetrix GeneChip HG-U95Av2 to analyze the temporal gene expression profile. Out of 12,625 genes present on the HG-U95Av2 GeneChip, expression of 5,432 genes was detected. There were 1,475 of 5,432 genes that displayed the differential expression compared to baseline (0 h). Fifty-four genes were upregulated (=<twofold) while 61 genes were downregulated (>=twofold) at 24-48 h after the Sim treatment. Many new target genes and pathways modulated by Sim were uncovered. This study indicates that many aspects of the pleiotropic effect of Sim on the endothelial cell function can be mediated by transcriptional control. Physiological function of 22% of 115 differentially expressed genes in Sim-treated HCAEC are currently unknown. These newly identified genes could be useful for new mechanistic study and new therapeutic modalities. Expressions of 13 out of 18 genes (>70%) in the cell cycle/proliferation control process were significantly inhibited by the Sim treatment. CDC25B and ITGB4 gene expressions were validated by RT-PCR and Western blotting. Sim's inhibitory effect of on HCAEC growth was confirmed by the measurement of [3H]thymidine incorporation into the DNA synthesis. Further indepth analysis of this effect may shed light on molecular mechanisms of Sim's beneficial inhibition of neointima formation in the atherosclerotic artery stenosis.
Key words: Simvastatin; Coronary artery endothelial cells; Microarray; Gene expression
Address correspondence to Shui Qing Ye, M.D., Ph.D., Department of Surgery and Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Medical Science Bldg. #M701, DC097.00, One Hospital Drive, Columbia, MO 65212, USA. E-mail: firstname.lastname@example.org
The Role of Neurotrophins During Early Development
Department of Anatomy and Cell Biology and Department of Otolaryngology, State University of New York, Brooklyn, NY, USA
The effects of neurotrophins during the middle and late stages of development are well known. It was previously thought that neurotrophins had no role during early development, but this is not the case and is the subject of this review article. The earliest neurotrophin receptor expressed is that for neurotrophin-3 (NT-3). TrkC is detected in the neural plate and is present in the neural tube. Initially, the distribution of TrkC is homogenous, but it becomes localized to specific regions of the neural tube as the neural tube differentiates. The receptor for brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5), TrkB, is detected somewhat later than TrkC in the neural tube where it is also differentially localized. In contrast, the NGF receptor, TrkA, was not detected during early development. Both NT-3 and BDNF have been shown to have effects in vitro during early development. NT-3 caused an increase in neurite outgrowth and apoptosis in neural plate explants, and promoted differentiation of progenitors into motoneurons. BDNF increased the number of motoneurons in neural tube explants. These data suggest that NT-3 and BDNF may play a role during early development in vivo.
Key words: Neurotrophin; Trk; p75; Nerve growth factor; Brain-derived neurotrophic factor; Neurulation; Neural plate; Neural tube; Rhombomere;
Address correspondence to Paulette Bernd, at her present address: Professor of Clinical Pathology & Cell Biology, Columbia University Physicians and Surgeons, 630 West 168 Street, New York, NY 10032, USA. Tel: (212) 305-7572; Fax: (212) 305-6595; E-mail: email@example.com