Award Date

1-1-2001

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Science

First Committee Member

George Plopper

Number of Pages

145

Abstract

Integrins are cellular adhesion receptors whose functions are critical to the progression of solid tissue cancers. The research described here involves four projects aimed at understanding how integrins modulate key elements of carcinogenesis, (adhesion and migration), on laminin basal lamina proteins; In order to carry out these studies, a novel fluorescence based protocol for quantifying cell migration towards a bound substrate was developed (Chapter 2). This method is more reliable, efficient, and informative than currently available protocols. I demonstrate its use as a screen for migration-blocking chemotherapeutic drugs; The small heat shock protein 27 (HSP27) is found to be over-expressed in many mammary carcinomas and can be an effective prognosticator of disease recurrence following treatment. The mechanisms for this action of HSP27 are not fully understood. In chapter 3, I demonstrate that over-expressed and activated HSP27 enhances migration towards laminin-5 in breast carcinoma cells through an intracellular signaling pathway involving mitogen activated protein kinase (MAPK/ERK 1/2). In addition, non-activated HSP27 in the same cells confers resistance to a subset of drugs that would halt migration; In a screen of drugs that block the function of specific intracellular signaling molecules, it was found that an inhibitor of G-protein mediated signaling blocked the integrin induced migration of breast epithelial cells on laminin-5. I demonstrated in chapter 4 that pro-migratory signaling cascades stimulated by the beta1 class of integrins are inhibited specifically by interruption of Galphai and not Galphas heterotrimeric G-protein isoforms. Furthermore, beta1 stimulation caused an increase of an intracellular G-protein effector (cAMP). Artificial manipulation of CAMP mimicked the effects of beta1 stimulation on migration of these cells. These data suggested for the first time that G-proteins function in integrin stimulated signal cascades; Because most cells express several types of integrins simultaneously, in chapter 5 I constructed a model whereby the contribution of individual integrin types to the net effect of integrin engagement could be analyzed. For this I demonstrated that intracellular calcium flux can be used as a reliable downstream marker of integrin stimulated signaling cascades, and that laminin-1 is a good model substrate for stimulating a variety of integrin subtypes. Next I describe the production and testing of six recombinant fragments of laminin-1 for the purpose of producing activating ligands of specific laminin-1 binding integrins.

Keywords

Adhesion; Analysis; Adhesion; Cancer; Integrin; Laminin; Mediated; Migration; Signaling; Vitro

Controlled Subject

Molecular biology; Cellular biology

File Format

pdf

File Size

3624.96 KB

Degree Grantor

University of Nevada, Las Vegas

Language

English

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