Title

Role of alpha5beta1 integrin in anchorage-dependent growth and TGFbeta-induced anchorage-independent growth

Date of Award

1998

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Molecular Cell and Developmental Biology

First Committee Member

Richard Assoian, Committee Chair

Abstract

Increasing evidence indicates the importance of adhesion in cell growth. In particular, alpha5beta1 integrin is implicated in regulating proliferation in normal and transformed cells. In the first part of this study, we examined the effects of increased or decreased expression of alpha5beta1 integrins in adherent NIH3T3 or NRK fibroblasts. A fourfold overexpression of alpha5beta1 integrin achieved by transfecting human alpha5-pECE cDNA into NIH3T3 fibroblasts resulted in no noticeable effects on cell cycle progression. However, a four-fold decrease in surface expression of this receptor accomplished by transfection of NRK fibroblasts with alpha5-antisense cDNA showed a 3--4 fold reduction in the proliferation rates. These cells appeared less spread, and staining of the actin filaments revealed poor defined stress fibers. Furthermore, seeding of alpha5-antisesense NRK cells on collagen, which completely restored cell spreading, failed to rescue cell proliferation, suggesting that the observed reduction in growth was a specific result of reduced alpha5beta1 integrin expression rather than inhibition of cell spreading. When cultured for prolonged periods of time, the alpha5-antisense NRK cells reverted to the original phenotype. Fast dividing clones outcompeted the slow growing transfectants, resulting in a new population expressing normal alpha5beta1 integrin levels. The analysis of cell cycle progression linked the alpha5beta1 integrin to a decreased cyclin A expression.In the second part of the study, we examined the effects of a reduced expression of alpha5beta1 integrin on TGFbeta-induced anchorage independent growth in NRK cells. Integrins become internalized when NRK cells are cultured in suspension, and TGFbeta induced anchorage-independent growth is preceded by restoration the surface expression of alpha5beta1 integrin. However, in alpha5-antisense NRK fibroblasts, TGFbeta was unable to restore the surface expression of alpha5beta1 integrin or induce anchorage independent growth, directly implicating alpha5beta1-integrin in a signaling pathway by which TGFbeta induces anchorage-independence in NRK fibroblasts.

Keywords

Biology, Molecular; Biology, Cell

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:9934217