Summer Student Research Program
Project Description
FACULTY SPONSOR’S NAME AND DEGREE: Muriel W. Lambert, Ph.D.
PHONE: (973) 972-4405
DEPARTMENT AND INTERNAL MAILING ADDRESS: Department of Pathology and Laboratory Medicine, Medical Sciences Building, NJMS
E-MAIL:
PROJECT TITLE (200 Characters max):
Factors Affecting Stability of Nonerythroid Alpha Spectrin in Fanconi Anemia Cells
HYPOTHESIS:
There are reduced levels of the structural protein, nonerythroid a spectrin (aIISp), in cells from patients with Fanconi anemia. We have hypothesized that this is due to reduced stability of this protein in these cells. In normal human cells, stability of aIISp is related to the action of several proteins, two of which are calmodulin and calpain. Calpain cleaves aIISp and calmodulin is thought to play a role in stimulating the activity of calpain.. We hypothesize that in FA cells the activity of these proteins is increased and that interaction of one or more of the FA proteins with these proteins may be an important factor in regulating their activity.
PROJECT DESCRIPTION (Include design, methodology, data collection, techniques, data analysis to be employed and evaluation and interpretation methodology)
Fanconi anemia (FA) is an autosomal recessive, bone marrow failure disorder. It is characterized by pancytopenia progressing to aplastic anemia, myelodysplasia, a markedly increased incidence of acute myelogeneous leukemia and diverse congenital abnormalities, which include developmental defects, growth retardation and skeletal deformities. FA cells are particularly hypersensitive to the clastogenic and cytotoxic effects of DNA interstrand cross-linking agents. Cells from FA patients have been shown to have a defect in DNA repair, particularly repair of DNA interstrand cross-links. On the basis of this sensitivity to DNA interstrand cross-linking agents and the presence of a defect in ability to repair damage produced by these agents, it has been proposed that an underlying mechanism for this disorder involves a DNA repair defect.
We have isolated from the nucleus of normal human cells a protein complex involved in repair of DNA interstrand cross-links. We have identified the structural protein, nonerythroid a spectrin (aIISp), as a component of this protein complex and demonstrated that it binds directly to DNA containing an interstrand cross-link and plays a role in repair of this type of damage. Studies in our laboratory have demonstrated that cells from a number of FA complementation groups are deficient in aIISp. We have developed a model in which we have hypothesized that aIISp localizes to sites of damage after cells are treated with a DNA interstrand cross-linking agent and acts as a scaffold to aid in the recruitment of repair proteins to the site to damage. In FA cells, where there are decreased levels of aIISp, there is reduced recruitment of repair proteins to sites of damage and thus reduced levels of DNA repair in these cells.
Studies in our laboratory indicate that reduced levels of aIISp in FA cells are due to reduced stability of this protein. An important question is thus what factors are responsible for the reduced stability of aIISp in FA cells. We hypothesize that one or more of the FA proteins is involved in maintaining the stability of aIISp and that these proteins may do so by either binding directly to aIISp or to one of the proteins that is involved in the breakdown of aIISp (i.e., calmodulin and calpain). One mechanism for the cleavage of aIISp by calpain is thought to involve the stimulation of calpain activity by calmodulin. We have hypothesized that the binding of one or more FA proteins to calmodulin may inhibit its ability to stimulate calpain and inturn lead to reduced aIISp cleavage in the cell. In the present project, studies will be carried out to determine whether any of the FA proteins bind to calmodulin. Co-immunoprecipitations will be carried out using nuclear extracts from normal human cells. Anti-calmodulin will be bound to protein-A-coated agarose beads and the beads incubated with normal human cell nuclear extracts. Various FA antibodies will be used to determine whether any of the FA proteins co-immunoprecipitate with calmodulin using western blot analysis.
These studies should show whether any of the FA proteins bind to calmodulin. If they do, this could be an indication that in normal cells this binding interferes with the ability of calmodulin to stimulate calpain activity and lead to the breakdown of aIISp. In FA cells, where there is a deficiency in these FA proteins, this association between the FA proteins and calmodulin would be deficient. This in turn could lead to increased breakdown of aIISp in these cells and to the reduced DNA repair capability observed.
SPONSOR’S MOST RECENT PUBLICATIONS RELEVANT TO THIS RESEARCH:
McMahon LW, Sangerman J, Goodman SR, Kumaresan K, Lambert MW 2001. Human alpha spectrin II and the FANCA, FANCC, and FANCG proteins bind to DNA containing psoralen interstrand cross-links. Biochemistry, 40:7025-7034.
Kumaresan KR, Hwang M, Thelen MP, Lambert MW 2002. Contribution of XPF functional domains to the 5’ and 3’ incisions produced at the site of a psoralen interstrand cross-link. Biochemistry, 41:890-896.
Sridharan D, Brown M, Lambert WC, McMahon LW, Lambert MW 2003. Nonerythroid alpha II spectrin is required for recruitment of FANCA and XPF to nuclear foci induced by DNA interstrand cross-links. J Cell Science, 116:823-835.
Lefferts JA, Lambert MW 2003. Fanconi anemia cell lines deficient in aII spectrin express normal levels of aII spectrin mRNA. Biochem Biophys Res Comm, 307:510-515.
Sridharan DM, McMahon LW, Lambert MW 2006. aII spectrin interacts with five groups of functionally important proteins in the nucleus. Cell Biol Internat, 30:866-878.
Kumaresan, K, Sridharan, D, McMahon, L, Lambert MW 2007. Deficiency in incisions produced by XPF at the site of a DNA interstrand cross-link in Fanconi anemia cells. Biochemistry, 46:14359-14368.
IS THIS PROJECT SUPPORTED BY EXTRAMURAL FUNDS?
Yes X or No
(IF YES, PLEASE SUPPLY THE GRANTING AGENCY’S NAME)
NIH - NHLBI
THIS PROJECT IS: Clinical X Laboratory Behavioral Other
THIS PROJECT IS CANCER-RELATED: YES
THIS PROJECT IS HEART, LUNG & BLOOD- RELATED: YES
THIS PROJECT EMPLOYS RADIOISOTOPES: NO
THIS PROJECT INVOLVES THE USE OF ANIMALS ; NO
PENDING APPROVED IACUC PROTOCOL #
THIS PROJECT INVOLVES THE USE OF HUMAN SUBJECTS: NO
PENDING APPROVED IRB PROTOCOL # M
THIS PROJECT IS SUITABLE FOR:
UNDERGRADUATE STUDENTS: ENTERING FRESHMAN
SOPHMORES: MEDICAL STUDENTS X ALL STUDENTS
THIS PROJECT IS WORK-STUDY: Yes or No X
WHAT WILL THE STUDENT LEARN FROM THIS EXPERIENCE?
The student will learn some basic molecular and biochemical techniques. This will include several assays in which techniques demonstrating ways to measure protein-protein interactions will be used. My laboratory has weekly meetings in which we go over laboratory data and recent papers on FA. From these meetings, the student will learn more about the FA research ongoing in my laboratory and the recent advances begin made in this field. The student should also learn the importance of basic research, how this research can be applied to learning more about the underlying basis of a specific hematological disorder, and the potential relevance of this research to translational studies on FA.