The application of high density microarray for analysis of mitogenic signaling and cell-cycle in the adrenal

Angiotensin II (AII) binds to specific G-protein coupled receptors and is m itogenic in adrenal, liver epithelial, and vascular smooth muscle cells. Th e H295R human adrenocortical cell line, which expresses AII receptors predo minantly of the AT(1) subclass, proliferates in response to treatment with AII. The induction and maintenance of cellular proliferation involves a pre cisely coordinated induction of a variety of genes. As the human genome seq uencing projects near completion a variety of high throughput technologies have been developed in order to create dynamic displays of genomic response s. One high throughput method, the gridded cDNA microarray has been develop ed 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 DI gene, wh ich encodes the regulatory subunit of the cyclin D1-dependent kinase (CD1K) required for phosphorylation of the retinoblastoma protein (pRB), was indu ced by An. in H295R cells. Abundance of the cyclin DI gene is rate-limiting in G(1) phase progression of the cell-cycle in a variety of cell types. AI I induced cyclin D1 promoter activity through a c-Fos and c-Jun binding seq uence at -954 bp. The abundance of c-Fos within this complex was increased by All treatment. Analysis of ALT signaling in adrenal cells by cDNA microa rray demonstrated an induction of the human homologue of Xenopus XPMC2 (HXP MC2). 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 DI and XPMC2H in All-induc ed cell-cycle progression and cellular proliferation in the adrenal.