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: mlambert@umdnj.edu
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  spectrin (IISp), 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 IISp is related to the action of several
proteins, two of which are calmodulin and calpain. Calpain cleaves IISp 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 crosslinking 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 
spectrin (IISp), 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 IISp. We have developed a model in which we have hypothesized that
IISp 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 IISp, there is reduced recruitment of repair
proteins to sites of damage and thus reduced levels of DNA repair in these cells.
Summer Student Research Program
Project Description
Studies in our laboratory indicate that reduced levels of IISp 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 IISp in FA cells. We hypothesize that one or more of the FA proteins is
involved in maintaining the stability of IISp and that these proteins may do so by either binding
directly to IISp or to one of the proteins that is involved in the breakdown of IISp (i.e.,
calmodulin and calpain). One mechanism for the cleavage of IISp 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 IISp 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 coimmunoprecipitate 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 IISp. 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 IISp 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 II spectrin express
normal levels of II spectrin mRNA. Biochem Biophys Res Comm, 307:510-515.
Sridharan DM, McMahon LW, Lambert MW 2006. II 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)
Summer Student Research Program
Project Description
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.