Recursos Técnicos - Posters Científicos
A Homogeneous FRET-based HTS Assay for quantification of pRb in Breast Cancer Cell Lines to monitor inhibition of GO / G1 cell phase transitionDescargar
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September 23, 2019
Breast cancer remains a global concern given the significant number of new cases reported annually. Recent advances in understanding the underlying biology of disease progression at the molecular level have proved beneficial for identification of new targets and agents against tumor cell growth. A key factor of cancer in general is a disruption of cell cycle control leading to unobstructed cell proliferation. The ability to identify control pathways and checkpoints to target may provide points of intervention through novel therapeutics. During normal cell cycle control the distinct phases of the cell cycle are conserved progressing from GO (quiescence) followed by G1 (pre-DNA sysnthesis). S (DNA synthesis), G2 (pre-division), and M (cell division). Of those, the progression from G1 to S provides a sentry point that restricts cell proliferation via the interaction between the cyclin-dependent kinases (CDKs) and cyclin proteins. One key role of a subgroup of CDKs, serine/threonine kinases, is the hyperphosphorylation of the retinoblastoma (Rb) gene product, pRb, in early G1 by CDK4 and CDK6 interacting with cyclin D1 resulting in inactivation and the subsequent release of a number of transcription factors necessary for passage into S phase. Previous observations suggest that CDK4/6 inhibition may prevent tumor growth and may help return cells to a near normal phenotype. Demonstrated here is a novel HTRF cell-based assay that simply and accurately quantifies endogenous phosphorylated retinoblastoma protein at Ser807/811 as a readout of the GO / G1 cell phase transition. The dose response and IC50 concentration was determined for a representative kinase inhibitor in a high throughput 384-well, homogeneous cell based assay format using several cell lines.