Jack A. Roth

p53 Suppressor Gene

1. The Role of p53 in Cancer.- 2. Gene Structure.- 3. Wild-Type versus Mutant p53.- 4. Biophysical and Biochemical Properties of the p53 Protein.- 5. Regulation and Modulation of the Function of p53.- 6. Potential Clinical Significance of the p53 Tumor Suppressor Gene in Cancer Patients.

High Throughput Profiling of Serum Phosphoproteins/Peptides Using the SELDI-TOF-MS Platform

Protein phosphorylation is a dynamic post-translational modification that plays a critical role in the regulation of a wide spectrum of biological events and cellular functions including signal transduction, gene expression, cell proliferation, and apoptosis. Determination of the sites and magnitudes of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. A high throughput analysis of phosphorylation of proteins would provide a simple, logical, and useful tool for a functional dissection and prediction of biological functions and signaling pathways in association with these important molecular events. We have developed a functional proteomics technique using the ProteinChip array-based SELDI-TOF-MS analysis for high throughput profiling of phosphoproteins/phosphopeptides in human serum for the early detection and diagnosis as well as for the molecular staging of human cancer. The methodology and experimental approach consists of five steps: (1) generation of a total peptide pool of serum proteins by a global trypsin digestion; (2) rapid isolation of phosphopeptides from the total serum peptide pool by an affinity selection, purification, and enrichment using a novel automated micro-bioprocessing system with phospho-antibody-conjugated paramagnetic beads and a hybrid magnet plate; (3) high throughput phosphopeptide analysis on ProteinChip arrays by automated SELDI-TOF-MS; and (4) bioinformatics and statistical methods for data analysis. This method with appropriate modifications may be equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins and for selectively isolating, profiling, and identifying phosphopeptides present in a highly complex phosphor-peptide mixture prepared from various human specimens such as cells, tissue samples, and serum and other body fluids.

Wild-Type versus Mutant p53

What is now known as p53 was initially identified as a normal cellular protein bound to SV40 large T antigen.1,2 Immunoprecipitation of large T antigen from a transformed mouse cell line coprecipitated a nuclear phosphoprotein of 53,000 molecular weight, hence called p53. The human p53 protein is composed of 393 amino acids and is located in the nucleus. p53 is present in all tissues but in such low quantities3–5 that it is difficult to detect by immunohistochemical techniques. On the other hand, the p53 protein has been detected at much higher levels in a large number of sporadic tumors and virally and chemically-transformed cell lines from mice and humans.6,7 Isolation and characterization of the p53 gene followed by early transfection studies indicated that p53 is capable of immortalizing primary rat embryonic fibroblast cells in culture. It was also found that p53 could cooperate with activated ras oncogene in cellular transformation of primary cells in culture.8–10

Analysis of Protein–Protein Interaction Using ProteinChip Array-Based SELDI-TOF Mass Spectrometry

Protein-protein interactions are key elements in the assembly of cellular regulatory and signaling protein complexes that integrate and transmit signals and information in controlling and regulating various cellular processes and functions. Many conventional methods of studying protein-protein interaction, such as the immuno-precipitation and immuno-blotting assay and the affinity-column pull-down and chromatographic analysis, are very time-consuming and labor intensive and lack accuracy and sensitivity. We have developed a simple, rapid, and sensitive assay using a ProteinChip array and SELDI-TOF mass spectrometry to analyze protein-protein interactions and map the crucial elements that are directly involved in these interactions. First, a purified bait protein or a synthetic peptide of interest is immobilized onto the preactivated surface of a PS10 or PS20 ProteinChip and the unoccupied surfaces on the chip are protected by application of a layer ethanolamine to prevent them from binding to other non-interactive proteins. Then, the target-containing cellular protein lysate or synthetic peptide containing the predicted amino acid sequence of protein-interaction motif is applied to the protected array with immobilized bait protein/peptide. The nonspecific proteins/peptides are washed off under various stringent conditions and only the proteins specifically interacting with the bait protein/peptide remain on the chip. Last, the captured interacting protein/peptide complexes are then analyzed by SELDI-TOF mass spectrometry and their identities are confirmed by their predicted distinctive masses. This method can be used to unambiguously detect the specific protein-protein interaction of known proteins/peptides, to easily identify potential cellular targets of proteins of interest, and to accurately analyze and map the structural elements of a given protein and its target proteins using synthetic peptides with the predicted potential protein interaction motifs.

Gene-Based Therapies for Lung Cancer

Recent advances in genetics, molecular biology, molecular pharmacology, and biomolecular technology have brought targeted therapeutic opportunities to the forefront of clinical development. Physician and patient communities are highly attracted to lung cancer management opportunities that may involve a personalized approach based on utilizing a unique cancer signal with a target-specific therapy. In this chapter, we will review several advanced clinical developments involving gene-based targeted therapies in lung cancer. Discussion will focus on replacement therapies for abnormal p53 function, FUS1 mediated molecular therapy, antisense technologies, and early developments with RNA interference technology.

Methods for Cancer Gene Therapy Using Tumor Suppressor Genes

Under experimental conditions, fusion of normal and malignant cells in many different combinations most often results in the suppression of the tumorigenic phenotype of tumor cells (1). This phenomenon led to a hypothesis that the normal genome might contain recessive cancer genes that, when expressed, would suppress the growth of tumors (2). Since the rdentification and cloning of the retinoblastoma (Rb) and p53 genes, the study of what are now called tumor suppressor genes has progressed rapidly. Although tumors generally develop through multiple changes in several genes, the malignant phenotype can be reversed by the introduction of a single chromosome derived from a normal cell, suggesting that single suppressor genes may be able to overcome the effects of multiple changes related to tumor progression (3).

2-Methoxyestradiol induziert p53 unabhängige Apoptosis beim Pankreaskarzinom und reduziert das Wachstum von Lungenmetastasen.

Das Pankreaskarzinom ist die funft haufigste Todesursache durch maligne Tumoren. Die 5-Jahres Uberlebensrate uberschreitet kaum 20% [1]. Neue Therapieansatze scheinen daher von grosem Interesse zu sein. 2-Methoxyestradiol (2-ME) ist ein naturlicher Ostrogenmetabolit, der endotheliales Zellwachstum und Angiogenese hemmen kann [2]. Wir fragten in unserer Studie, ob 2-ME das Wachstum des Pankreaskarzinoms hemmen kann und untersuchten den Effekt und Mechanismus. Die Ergebnisse zeigen, das 2-ME sowohl in vitro, als auch in vivo das Wachstum des Pankreaskarzinoms hemmt.

The Role of p53 in Cancer

The product of the p53 tumor suppressor gene was first identified as a tumor antigen that bound to simian virus 40 (SV40) T antigen and adenovirus E1B oncoproteins.1–3 The p53 protein originally was believed to have an oncogenic rather than a tumor suppressor function, since it could immortalize cells in culture4 and cooperate with the activated ras oncogene to transform cells in culture.5’6 Overexpression of p53 also enhanced the transformed phenotype of tumor cells.7

Regulation and Modulation of the Function of p53

The p53 protein has a structure reminiscent of other factors involved in transcription.1–5 As discussed previously, strong evidence that wild-type p53 functions as a regulator of transcription has come from biochemical and biological studies demonstrating that the protein can repress the interleukin-6 (IL-6) gene and stimulate the expression of the mdm2, gadd45, and waf1/cip1 growth-regulatory genes, whose promoters contain a p53-binding site. Proteins may bind to p53 and regulate its DNA-binding and transcription activities in a fashion similar to that observed for other transcription factors, such as those belonging to the TFII,3,6–13 the AP-1,14–19 and the myc and max20,21 factor families.

Biophysical and Biochemical Properties of the p53 Protein

The biochemical mechanism of p53 in the control of cell growth is not completely understood. Considerable evidence implicates regulation of gene transcription as a mechanism of p53 action in controlling cell growth. The protein does resemble a transcription factor1–5 in that it has an acidic domain that can transactivate reporter genesG6–12 and a basic carboxyl terminal domain that can bind nonspecifically to DNA13 (Fig. 4.1).