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The application of high density microarray for analysis of mitogenic signaling and cell-cycle in the adrenal
C Wang1, R Francis, S Harirchian
1The Albert Einstein Cancer Center, Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Abstract:
Angiotensin II (AII) binds to specific G-protein coupled receptors and is mitogenic in adrenal, liver epithelial, and vascular smooth muscle cells. The H295R human adrenocortical cell line, which expresses AII receptors predominantly of the AT1 subclass, proliferates in response to treatment with AII. The induction and maintenance of cellular proliferation involves a precisely coordinated induction of a variety of genes. As the human genome sequencing projects near completion a variety of high throughput technologies have been developed in order to create dynamic displays of genomic responses. One high throughput method, the gridded cDNA microarray has been developed in which immobilised DNA samples are hybridized on glass slides for the identification of global genomic responses. For this purpose high precision robotic microarrayers have been developed at AECOM. The cyclin D1 gene, which encodes the regulatory subunit of the cyclin D1-dependent kinase (CD1K) required for phosphorylation of the retinoblastoma protein (pRB), was induced by AII in H295R cells. Abundance of the cyclin D1 gene is rate-limiting in G1 phase progression of the cell-cycle in a variety of cell types. AII induced cyclin D1 promoter activity through a c-Fos and c-Jun binding sequence at -954 bp. Theabundance of c-Fos within this complex was increased by AII treatment. Analysis of AII signaling in adrenal cells by cDNA microarray demonstrated an induction of the human homologue of Xenopus XPMC2 (HXPMC2). The cDNA for XPMC2 was previously shown to rescue mitotic catastrophe in mutant S. Pombe defective in cdc2 kinase function. Further studies are required to determine the requirement for cyclin D1 and XPMC2H in AII-induced cell-cycle progression and cellular proliferation in the adrenal.
Insights
Angiotensin II (AII) stimulates adrenal cell proliferation by upregulating cyclin D1 and HXPMC2 genes. These findings reveal key molecular players in AII-induced cell growth and cell-cycle progression.
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Biology
Background:
- Angiotensin II (AII) is a mitogen for various cell types, including adrenal cells, acting through G-protein coupled receptors.
- The H295R human adrenocortical cell line expresses AT1 receptors and proliferates upon AII stimulation.
- Cellular proliferation is a complex process involving coordinated gene induction.
Purpose of the Study:
- To investigate the molecular mechanisms underlying Angiotensin II-induced proliferation in H295R adrenocortical cells.
- To identify genes regulated by AII signaling pathways involved in cell-cycle progression.
Main Methods:
- Utilized the H295R human adrenocortical cell line.
- Employed high-throughput cDNA microarray technology for global gene expression analysis.
- Investigated gene promoter activity and protein complex formation.
Main Results:
- AII treatment induced the expression of the cyclin D1 gene, a key regulator of cell-cycle progression.
- AII enhanced cyclin D1 promoter activity via a c-Fos and c-Jun binding site.
- cDNA microarray analysis revealed AII-induced expression of the human homologue of Xenopus XPMC2 (HXPMC2).
Conclusions:
- Cyclin D1 and HXPMC2 are induced by Angiotensin II in H295R cells.
- These genes play a role in AII-induced cell-cycle progression and proliferation in adrenal cells.
- Further research is needed to fully elucidate the roles of cyclin D1 and HXPMC2 in this process.