Doctoral Dissertations

Date of Award

8-2001

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Tuan Vo-Dinh

Committee Members

Jeffery Becker, Stephen Kennel, Stephen Oliver

Abstract

In early 1996, the Fragile Histidine Triad (FHIT) gene was cloned and shown to straddle the most active of the fragile human chromosome sites at chromosome band 3pl4.2. The exceptionally large FHIT locus encompasses a hereditary renal carcinoma-associated chromosome translocation breakpoint. The size of the FHIT gene is about 1 megabase of genomic DNA, encoding a 1.1 kb niRNA message and a 16.8 kDa protein with diadenosine triphosphate hydrolase activity.

Early studies of a number of important human tumors such as breast, gastric, renal, and lung carcinoma and pancreatic adenocarcinoma have revealed that FHIT RNA expression was frequently altered and these alterations correlated with deletions in the FHIT gene, suggesting a role for this gene in development of cancer. There has also been a correlation between complete absence of Fhit protein and the early clinical stages of cancer. Such observations implicated FHIT as a putative tumor suppressor gene. Nonetheless, several lines of evidence have called into question the role of FHIT as a "classical" tumor suppressor gene, and raised the question of whether its apparent involvement simply reflects its location within an unstable region of the genome. Observation of biallelic deletions rather than mutations of the FHIT gene in cancers prompted a number of investigators to reject FHIT as a suppressor gene. In addition, consistent effects of exogenous FHIT on growth in cultures had not been observed. Additionally, experiments transfecting wild-type (wi) FHIT into tumor cell lines with FHIT abnormalities have produced conflicting results regarding suppression of tumorigenesis in vivo. The primary objective of this project was to investigate whether the FHIT gene was indeed a tumor suppressor gene. The goal was to establish a representation of the underlying molecular and cellular mechanisms of action of FHIT gene in suppression of tumorigenesis. The results demonstrate that the FHIT gene is indeed a tumor suppressor gene and that Fhit expression plays a possible role in induction of apoptosis. Our data indicates that Fhit protein alters the mitochondrial flux and efflux of molecules causing alterations in the transmembrane potential in the presence of apoptotic stimuli. We observed that induction of apoptosis in cells expressing the Fhit protein also involved the release of mitochondrial cytochrome c from the mitochondria and its subsequent translocation into the cytoplasmic compartment.

This investigation was also aimed at developing and applying novel spectroscopic and biosensing techniques and protocols designed to provide alternative methods for gene and protein identification, and defining gene function at the cellular and molecular levels for applications in biological research and clinical diagnosis laboratories. For the design of these systems, we took advantage of optical spectroscopy techniques including fluorescence, and synchronous luminescence spectroscopy, the biochip technology, and various microscopy methods. These modern technologies, developed at ORNL, have the potential to be selective, as well as sensitive, in providing information to understand how gene expression impacts a specific biological system.

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