Identificação e caracterização de biomarcadores teciduais e sorológicos no câncer de mama por Phage Display

by de Sousa, Cristina Soares

Abstract (Summary)
CHAPTER II: Breast cancer is one of the main causes of death among women, as thereis no primary prevention. Early detection is the main objective aiming decreasemortality and increase survival. We used phage display technology to isolateligand peptides to breast cancer tissues in order to select potential biomarkers forthe improvement of diagnosis and treatment. Two random peptide libraries withseven and twelve residues expressed in fusion with the pIII protein of the M13bacteriophage, Ph.D.-7 and Ph.D.-12, respectively, were firstly submitted to a preclearing by placing them in contact with proteins of normal breast tissues, in oderto eliminate peptides that may recognize healthy tissues. Remaining phages in thesupernatant was further selected through three rounds of positive selection. DNAof selected phage were sequenced and translated. Peptide sequences were thensubmitted to bioinformatic analyzes, ELISA, Dot-blotting, western-blotting andimmunohistochemistry assays to confirm the selection strategy and to providefurther evidences of the putative biological targets. The immunohistochemistryanalysis was performed with six selected phage (BC04, BC05, BC07, BC11,BC12, BC17) in breast cancer, and a mixture of three phage (BC11, BC12, BC17)has been tested in breast and ovary cancers, non-Hodgkin lymphoma, andmelanoma. All phages presented high information content with significant ELISAindexes. The Dot-blotting and western-blotting assays have validated the selectionby demonstrating high specificity to tumor proteins. The immunohistochemistryanalyzes have shown specific binding only to tumor cells. The mixture of phageshas recognized all types of tumor tissues, except melanoma. Three selectedphage, BC04, BC05 and BC07 have specifically marked the vascular walls oftumor cells. The selected phage may be used in the future as tissue biomarkersfor diagnosis and therapeutic procedures in breast cancer. The high informationcontent and the significant data presented by all immunological assays havevalidated the Phage Display subtractive selection strategy. CHAPTER III: Breast cancer is the most frequent malignant pathology in the Brazilianwomen population, which is aggravated by diagnosis in later stages of thedisease. The aim of this investigation was to select and identify ligand peptides toserum proteins of women with breast cancer through phage display in order to usethem as serological biomarkers. Selection of phages was performed in three stepsusing a random peptide library of 12 residues (Ph.D.-12). The first two selectionsteps consisted of a pre clearing using the phage libraries against sera proteins ofhealthy individuals, followed by a second selection against sera proteins ofpatients with benign breast diseases. After subtraction, the library was submittedto a positive selection against sera of patients with breast cancer. Phage ligandswere selected, amplified, and the DNA was sequenced and translated. Peptidesequences were subimtted bioinformatic analyses and to ELISA and dot-blottingassays to confirm selection success. All clones presented significant ELISAindexes and a high information content. The sequence IETYESTHQSPP was themost frequent and presented a high reactivity against IgG from breast cancerpatients´ in the dot-blotting analysis. Clones T03, T18 and T09 presented similaritywith the ubiquitin and clones T06, T19, T04, T10, T11, T14, T17 with zinc fingermotifs. The fact of different clones to present similarity for the same type of proteinsuggests that different motifs of one same protein can be epitopes recognized byauto-antibodies or that these motifs are interacting with proteins associated tobreast cancer, since biopanning was made with total serum.
This document abstract is also available in Portuguese.
Bibliographical Information:

Advisor:Luiz Ricardo Goulart Filho; Marco Antonio Arap; Veridiana de Melo Rodrigues Ávila; Warwick Estevam Kerr; José Cláudio Casali da Rocha; Ana Maria Bonetti

School:Universidade Federal de Uberlândia

School Location:Brazil

Source Type:Master's Thesis

Keywords:Breast cancer Phage Display


Date of Publication:02/16/2007

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