Anti-MCL1 monoclonal antibody, clone 21G7

Short half-life proteins regulate many essential processes, including cell cycle, transcription, and apoptosis. However, few well-characterized protein-turnover pathways have been identified because traditional methods to measure protein half-life are time and labor intensive. To overcome this barrier, we developed a protein stability probe and high-content screening pipeline for novel regulators of short half-life proteins using automated image analysis. Our pilot probe consists of the short half-life protein c-MYC (MYC) fused to Venus fluorescent protein (MYC-Venus). This probe enables protein half-life to be scored as a function of fluorescence intensity and distribution. Rapid turnover prevents maximal fluorescence of the probe due to the relatively longer maturation time of the fluorescent protein. Cells expressing the MYC-Venus probe were analyzed using a pipeline in which automated confocal microscopy and image analyses were used to score MYC-Venus stability by two strategies: assaying the percentage of cells with Venus fluorescence above background, and phenotypic comparative analysis. To evaluate this high-content screening pipeline and our probe, a kinase inhibitor library was screened by confocal microscopy to identify known and novel kinases that regulate MYC stability. Compounds identified were shown to increase the half-life of both MYC-Venus and endogenous MYC, validating the probe and pipeline. Fusion of another short half-life protein, myeloid cell leukemia 1 (MCL1), with Venus also demonstrated an increase in percent Venus-positive cells after treatment with inhibitors known to stabilize MCL1. Together, the results validate the use of our automated microscopy and image analysis pipeline of stability probe-expressing cells to rapidly and quantitatively identify regulators of short half-life proteins. (c) 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Patients with urothelial carcinoma frequently fail to respond to first-line chemotherapy using cisplatin and gemcitabine due to development of resistant tumor cells. The aim of the present study was to investigate whether an alternative treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that induces tumor cell death via the extrinsic apoptotic pathway may be effective against chemotherapy-resistant urothelial cancer cell lines. The viability of the urothelial cancer cell line RT112 and its chemotherapy-adapted sublines was investigated by MTT assay. The expression of anti-apoptotic proteins was determined by western blotting and the individual roles of cellular inhibitor of apoptosis protein (cIAP)1, cIAP2, x-linked inhibitor of apoptosis protein (XIAP) and induced myeloid leukemia cell differentiation protein (Mcl-1) were investigated by siRNA-mediated depletion. In particular, the bladder cancer sublines that were resistant to gemcitabine and cisplatin were cross-resistant to TRAIL. Resistant cells displayed upregulation of anti-apoptotic molecules compared with the parental cell line. Treatment with the second mitochondrial activator of caspases (SMAC) mimetic LCL-161 that antagonizes cIAP1, cIAP2 and XIAP resensitized chemoresistant cells to TRAIL. The resensitization of tumor cells to TRAIL was confirmed by depletion of antiapoptotic proteins with siRNA. Collectively, the findings of the present study demonstrated that SMAC mimetic LCL-161 increased the sensitivity of the parental cell line RT112 and chemotherapy-resistant sublines to TRAIL, suggesting that inhibiting anti-apoptotic molecules renders TRAIL therapy highly effective for chemotherapy-sensitive and -resistant urothelial cancer cells.