pervanadate and Colorectal-Neoplasms

In tumor cells, high phosphorylation levels of receptor tyrosine kinases may occur in the absence of exogenous ligands due to autocrine signaling or enhanced tyrosine kinase activity. Here we show that the phosphorylation state of the endogenous epidermal growth factor receptor (EGFR) can be quantitatively imaged in tumor cells and tissues by detecting fluorescence resonance energy transfer between fluorophores conjugated to antibodies against the receptor and phosphotyrosine, respectively. Five different human colorectal cell lines were analyzed for activity and expression of EGFR. All cell lines exhibited basal EGFR phosphorylation under serum starvation conditions. Phosphorylation levels increased after stimulation with EGF or pervanadate, dependent on the level of basal EGFR phosphorylation in the respective cell lines. This basal activity correlated inversely with receptor expression. Using the acceptor photobleaching fluorescence resonance energy transfer imaging approach, a significantly higher phosphorylation state of EGFR was also found in resected human colorectal tumor samples as compared with adjacent healthy tissue. Imaging of EGFR phosphorylation may thus serve as a valuable tool to investigate the role of receptor tyrosine kinase activity in malignant cell growth.

Topics: Cell Line, Tumor; Cell Proliferation; Cloning, Molecular; Colorectal Neoplasms; Culture Media, Serum-Free; DNA, Complementary; Enzyme-Linked Immunosorbent Assay; ErbB Receptors; Fluorescence Resonance Energy Transfer; Humans; Immunoblotting; Immunoprecipitation; Kinetics; Microscopy, Confocal; Microscopy, Fluorescence; Phosphorylation; Phosphotyrosine; Receptor Protein-Tyrosine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA; Signal Transduction; Time Factors; Vanadates

Overexpression of the epidermal growth factor receptor (EGFR) and its increased tyrosine kinase activity are implicated in colorectal cancer (CRC) development and malignant progression. The C57BL/6J-Min/+ (Min/+) mouse is a model for CRC and develops numerous intestinal adenomas. We analyzed the normal mucosa of Min/+ and Apc+/+ (WT) littermate mice together with Apc-null adenomas to gain insight into the roles of Egfr in these intestinal tissues. Protein analyses showed that Egfr activity was highest in the tumors, and also up-regulated in Min/+ relative to WT enterocytes. Expression of ubiquitylated Egfr (Egfr-Ub) was increased in Min/+ enterocytes and tumors. Tumors exhibited increased association of Egfr with clathrin heavy chain (CHC), Gab1, and p85alpha, the regulatory subunit of phosphoinositide 3-kinase (PI3K), and tumors also overexpressed c-Src, PDK1, and Akt. Immunohistochemistry for Akt-p-Ser473 revealed a low level of this active kinase in Min/+ and WT enterocytes and its strong presence in tumors. Prostaglandin E2 (PGE2) is a product of cyclooxygenase-2 (Cox-2) activity that is up-regulated in Min/+ tumors and transactivates Egfr. PGE2 expression was significantly higher in untreated Min/+ tumors and reduced by treatment with the Cox-2 inhibitor, celecoxib. Dietary administration of this NSAID also inhibited Egfr activity in tumors. Increased activation of the EGFR-PI3K-Akt signaling pathway in tumors relative to Apc+/+ and ApcMin/+ enterocytes provides potential opportunities for therapeutic interventions to differentially suppress tumor formation, promotion, progression, and/or recurrence.

Topics: Adaptor Proteins, Signal Transducing; Adenoma; Adenomatous Polyposis Coli Protein; Alleles; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Clathrin; Colorectal Neoplasms; Cyclooxygenase 2; Dinoprostone; Disease Progression; Enterocytes; Enzyme-Linked Immunosorbent Assay; ErbB Receptors; Genes, APC; HeLa Cells; Humans; Immunoblotting; Immunohistochemistry; Immunoprecipitation; Intestinal Mucosa; Intestine, Small; Isoenzymes; Jurkat Cells; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Models, Biological; Mutation; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Prostaglandin-Endoperoxide Synthases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Recurrence; Signal Transduction; src-Family Kinases; Tissue Distribution; Transcriptional Activation; Tyrosine; Ubiquitin; Up-Regulation; Vanadates