Abstract 5170: Novel discovery of P38 activation: Expression in A431 cells in in vitro and in vivo models of cancer, measured by a novel assay for p-P38 .

The p38 enzyme, also known as mitogen activated protein kinase or MAPK (pT180/pY182), is responsive to cellular stress such as osmotic shock, heat shock, and irradiation, and when activated, modulates the cell cycle, activation of DNA repair, regulation of protein translation and initiation of immune response. The p38 pathway consists of a cascade of protein kinases which ultimately leads to its phosphorylation on specific tyrosine and threonine residues. Growth factors play an important role in the activation of p38 MAPK. The binding of EGF or other specific growth factors to external receptors activate its kinase activity. The activation of p38 MAP kinase can directly influence gene transcription. A growing number of transcription factors are known to be direct targets of p38, thus its implication in many forms of cancer. In the A431 human epidermoid carcinoma cell line, proliferation and migration responses to extracellular stimuli require activation of distinct pathways. In this study we describe the use of the BioScale ViBE workstation and AMMP (acoustic membrane microparticle) assay technology to study the expression of p-p38 in lysates of A431 epidermoid carcinoma cells with or without stimulation of surface receptors for epidermal growth factor, compared to xenograft tumors of the same cell line. The AMMP p-p38 assay is robust and reproducible, with CVs less than 6% for within day and between day measurements. The assay demonstrates little interference from cell or tumor lysate matrices. Results from the p-p38 AMMP assays demonstrate that, in a group of ten A431 tumors, all were positive for the activated analyte. There was heterogeneity in the expression of the p-p38 in the tumors but all gave a positive assay response. Recombinant p-p38 was used as a positive control in each experiment. The results of the phospho-p38 AMMP assay were compared to western blots for the detection of p- p38 in EGF stimulated A431 cell lysates and untreated tumor lysates. The western blots were confirmation of presence or absence of the AMMP measured analyte in the samples. p-p38 was detected by western blot with similar intensity for equal protein loading of each sample type. The elucidation of p-p38 in A431 tumors in untreated nu/nu mice was unexpected as the EGF receptor was not activated based on AMMP results for P-EGFR derived from the same tumor sample set. This would indicate that the P38 was activated through a pathway other than the EGFR pathway. The observation of similar signals between the p-p38 levels detected between EGF stimulated A431 cells grown in vitro and in vivo tumors was also unexpected, again pointing to different pathways of activation. These results demonstrate that the BioScale AMMP assay for detection of p-p38 can be a powerful tool for the study of cancer, apoptosis or cell signalling, giving sensitive and reproducible results without interference from matrix components. Citation Format: Lee Anne Beausang, Kristen Leong, W. Matthew Dickerson, Ashley Saab, Edward M. Alderman. Novel discovery of P38 activation: Expression in A431 cells in in vitro and in vivo models of cancer, measured by a novel assay for p-P38 . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5170. doi:10.1158/1538-7445.AM2013-5170