Proteomic characterization of harvested pseudopodia with differential gel electrophoresis and specific antibodies

Abstract

Malignant gliomas (astrocytomas) are lethal tumors that invade the brain. Invasive cell migration is initiated by extension of pseudopodia into interstitial spaces. In this study, U87 glioma cells formed pseudopodia in vitro as cells pushed through 3 lm pores of polycarbonate membranes. Harvesting pseudopodia in a novel two-step method provided material for proteomic analysis. Differences in the protein profiles of pseudopodia and whole cells were found using differential gel electrophoresis (DIGE) and immunoblotting. Proteins from twodimensional (2D) gels with M R 's of 20-100 kDa and pI's of 3.0-10.0 were identified by peptide mass fingerprinting analysis using mass spectrometry. For DIGE, lysates of pseudopodia and whole cells were each labeled with electrophilic forms of fluorescent dyes, Cy3 or Cy5, and analyzed as mixtures. Analysis was repeated with reciprocal labeling. Differences in protein distributions were detected by manual inspection and computer analysis. Topographical digital maps of the scanned gels were used for algorithmic spot matching, normalization of background, quantifying spot differences, and elimination of artifacts. Pseudopodial proteins in Coomassie-stained 2D gels included isoforms of glycolytic enzymes as the largest group, seven of 24 proteins. Peptide mass fingerprint analysis of DIGE gels demonstrated increased isoforms of annexin (Anx) I, AnxII, enolase, pyruvate kinase, and aldolase, and decreased mitochondrial manganese superoxide dismutase and transketolase in pseudopodia. Specific antibodies showed restricted immunoreactivity of the hepatocyte growth factor (HGF) a chain to pseudopodia, indicating localization of its active form. Met (the HGF receptor), actin, and total AnxI were increased in pseudopodial lysates on immunoblots. Increased constituents of the pseudopodial proteome in glioma cells, identified in this study as actin, HGF, Met, and isoforms of AnxI, AnxII, and several glycolytic enzymes, represent therapeutic targets to consider for suppression of tumor invasion.

Tumor cells from ultrasonic aspirations of glioblastomas migrate and form spheres with radial outgrowth

“…Pseudopodia and unmigrated cells from the second specimen were stored at −80° C on glass slides and were later prepared as urea-based lysates in a procedure previously described [9]. Lysates were electrophoresed in a 10% gel under standard reducing conditions.…”

“…A method previously described for harvesting pseudopodia [9] was adapted for ultrasonically aspirated specimens. Cell suspensions were not filtered to remove tissue fragments to maximize the yield of pseudopodia.…”

Pseudopodial Actin Dynamics Control Epithelial-Mesenchymal Transition in Metastatic Cancer Cells

“…However, although EMT is closely associated with the formation of protrusive pseudopodial structures, the relationship between pseudopod formation and EMT has yet to be characterized. Molecular characterization of purified pseudopodial domains of fibroblasts, neurons, and cancer cells has led to the characterization of various populations of pseudopodenriched RNAs and proteins (16)(17)(18)(19)(20)(21). However, a comprehensive analysis of the mRNA and protein composition of pseudopodia of metastatic human cancer cells has yet to be undertaken.…”

Protrusion-localized STAT3 mRNA promotes metastasis of highly metastatic hepatocellular carcinoma cells in vitro

“…Several human cancer cell lines have been studied, using microarrays and proteomic analyses, to identify the specific proteins and transcripts present in the pseudopodia and involved in pseudopod protrusion, tumor cell migration and invasion [11] . However, a comprehensive analysis of the mRNAs and proteins localized in the protrusions of metastatic HCC cells and their function in the invasion and metastasis of these cells is still lacking [12][13][14] .…”