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ENHANCEMENT OF STABILITY OF NONERYTHROID ALPHA SPECTRIN IN FANCONI ANEMIA CELLS

by
Pan Zhang
Department of Pathology & Laboratory Medicine

M.D. 2001, ShanXi Medical University
M.S. 2004, Peking University


Thesis Advisor: Muriel W. Lambert, Ph.D.

Professor

Department of Pathology & Laboratory Medicine

Friday, September 18, 2009
10:00 a.m., MSB C-555


Abstract

Spectrin is a structural protein which is a central component of the cytoskeleton and plays an important role in providing support for the cell membrane and maintaining cell shape. It is also involved in organelle movement in the cell, protein sorting, neurotransmitter release and cell growth and differentiation. Our laboratory has demonstrated that nonerythroid á spectrin (áIISp) is present in mammalian cell nuclei where it plays a role in the repair of DNA interstrand crosslinks (ICL). It interacts with a number of proteins participating in ICL repair. These include XPF and a number of FA proteins. Our laboratory has proposed that one function of áIISp in the nucleus is to act as a scaffold where it aids in recruiting repair proteins to the site of the ICL. Our laboratory has shown that knockdown of áIISp in normal human cells by siRNA leads to cellular hypersensitivity to ICL agents and increased chromosome instability, this further strengthens the view that áIISp is important in ICL repair.
Fanconi anemia (FA) is a rare genetic disease, characterized by spontaneous bone marrow failure, a predisposition to development of cancer and various developmental abnormalities. There are 13 complementation groups of FA and cells from each of these groups are defective in ICL repair. In addition to a deficiency in specific FA proteins in cells from each of these complementation groups, there is also a deficiency in áIISp. This deficiency is due to breakdown of áIISp in these cells and our laboratory has proposed that FA proteins play a role in maintaining áIISp stability. The goals of the present study were to examine potential mechanisms for the increased breakdown of áIISp in FA cells and to determine whether some of the cellular phenotypic characteristics of FA, such as increased hypersensitivity to ICL agents and decreased DNA repair, could be reversed by restoring levels of áIISp to normal in these cells.
Cells from a number of FA complementation groups were examined for levels and activity of calpain, a protease which cleaves áIISp. The results show that increased calpain activity was found in FA-A, FA-C, FA-D2, FA-F, and FA-G cells compared to normal cells. This correlated with decreased levels of áIISp in these cells. µ-calpain levels, however, were similar to those in normal cells. Correction of these FA cells with the corresponding FA cDNA resulted in reduction in the levels of calpain activity to those found in normal cells. These findings indicate that in FA cells increased µ-calpain activity can account for the increased cleavage of áIISp.
In order to determine whether any of the FA proteins interacted with µ-calpain, co-immunoprecipitation studies were undertaken. These studies showed that FANCA, FANCC, FANCD2, FANCF and FANCG co-immunoprecipitated with µ-calpain indicating that there was interaction between them. To determine whether this interaction was direct, yeast two-hybrid analysis was undertaken. These studies showed that FANCA and FANCG bound to µ-calpain directly, whereas, FANCC did not. These results indicate that in normal cells, FANCA and FANCG interact directly with µ-calpain and could thus potentially exert an inhibitory effect on µ-calpain activity. FANCC and possibly other FA proteins could interact with µ-calpain indirectly via binding to FANCA or FANCG and thus facilitate the interactions of FANCA and FANCG with µ-calpain. In FA cells, in the absence of FA proteins, the inhibition of µ-calpain activity could be deficient, leading to increased µ-calpain activity in these cells.
Studies were then carried out to determine whether reduction in levels of µ-calpain in FA-A cells by siRNA could lead to restoration of áIISp in FA-A cells to normal levels and also reverse some of the cellular phenotypic defects observed in these cells. Forty-eight hours after transfection with µ-calpain siRNA, µ-calpain levels in FA-A cells were decreased to 44% of those found in non-target siRNA transfected FA-A cells. In the transfected cells, levels of áIISp returned to those found in normal cells. Cell survival data was also obtained on FA-A cells after µ-calpain knockdown. The results showed that after these cells were damaged with mitomycin C (MMC), an ICL agent, the µ-calpain siRNA transfected FA-A cells exhibited increased cell survival compared to non-target siRNA transfected FA-A cells. In addition, in order to determine if the µ-calpain siRNA transfected FA-A cells could function in ICL repair, indirect immunofluorescence studies were carried out. The results showed that after transfection of FA-A cells with µ-calpain siRNA, áIISp and XPF nuclear foci formed unlike the non-target siRNA transfected cells, in which there was a lack of foci formation. These data further strengthen the hypothesis that áIISp acts as a scaffold in the nucleus to recruit proteins involved in ICL repair and facilitate the repair process. The deficiency in áIISp in FA cells could contribute to the hypersensitivity of these cells to ICL agents and to the defect in ability to repair ICLs. Restoration of áIISp in FA-A cells by decreasing its breakdown by µ-calpain could thus aid in correcting the cellular phenotypic defects seen in these cells and thus provide a possible promising therapeutic target in the clinic in the future.


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