Programmed Cell-Death Mechanism Analysis Using Same-Cell, Multimode DNA and Proteoform Electrophoresis

Abstract

Gaining insight into the timing of cell apoptosis events requires single-cell-resolution measurements of cell viability. We explore the supposition that mechanism-based scrutiny of programmed cell death would benefit from same-cell analysis of both the DNA state (intact vs fragmented) and the protein states, specifically the full-length vs cleaved state of the DNA-repair protein PARP1, which is cleaved by caspase-3 during caspase-dependent apoptosis. To make this same-cell, multimode measurement, we introduce the single-cell electrophoresis-based viability and protein (SEVAP) assay. Using SEVAP, we (1) isolate human breast cancer SKBR3 cells in microwells molded in thin polyacrylamide gels, (2) electrophoretically separate protein molecular states and DNA molecular states—using differences in electrophoretic mobility—from each single-cell lysate, and (3) perform in-gel DNA staining and PARP1 immunoprobing. Performed in an open microfluidic device, SEVAP scrutinized hundreds to thousands of individual SKBR3 cells. In each single-cell lysate separation, SEVAP baseline-resolved fragmented DNA from intact DNA ( R s = 5.17) as well as cleaved PARP1 from full-length PARP1 ( R s = 0.66). Comparing apoptotic and viable cells showed statistically similar profiles (expression, mobility, peak width) of housekeeping protein β-tubulin (Mann–Whitney U test). Clustering and cross-correlation analysis of DNA migration and PARP1 migration identified nonapoptotic vs apoptotic cells. Clustering analysis further suggested that cleaved PARP1 is a suitable apoptosis marker for this system. SEVAP is an efficient, multimode, end-point assay designed to elucidate cell-to-cell heterogeneity in mechanism-specific signaling during programmed cell death.

Gels in Microscale Electrophoresis

“…The gel system includes polyacrylamide wells and buffer systems designed to minimize diffusion (74,75) or concentrate proteins (54) following cell lysis to release proteins into the separation channel or gel rinsing prior to immunoblotting (76). This strategy has been applied to high-throughput analyses of circulating human tumor cells (77), smooth aortic mouse cells (78), and cancer cells (38,79). The benefit of this approach is that it allows for quantitative studies of cellular heterogeneity that can be described in terms of relevant proteins that are indicative of or correlate with different chemical pathways.…”

“…Examples include motility (80), cellular differentiation (78), or mitogen-activated protein (MAP)-kinase activity (79). Additionally, the technique can be applied to simultaneously evaluate DNA cleavage and enzyme activity in the same gel, as was demonstrated for apoptosis (38). In other cases, RNA and DNA are collected after the protein detection and analyzed offline (81).…”

Microfluidic Compartmentalization Platforms for Single Cell Analysis

“…The following procedures are almost identical to the conventional Western blot, making the platform easily to apply. The device has helped investigate the effect of osmolarity on kinase phosphorylation [ 85 ] and identify the apoptotic state of cells [ 86 ].…”