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Hematology/Oncology

Kupfer Laboratory

The Kupfer lab works on the relationship of genomic instability and the propensity towards development of cancer. Specifically, we focus on the genetic syndrome Fanconi anemia (FA). Interestingly, children with FA are born with congenital anomalies and develop aplastic anemia and an assortment of leukemias and other cancers. FA serves as a paradigm where the disciplines of development, genetics, and molecular oncology come together. Like other cancer susceptibility syndromes, such as ataxia telangiectasia and xeroderma pigmentosum, FA patients exhibit a unique hypersensitivity to DNA crosslinking agents, which is the key to the biology of FA. Unlike the other syndromes, exceedingly little is known about FA. Eleven complementation groups have been elucidated, with all exhibiting similar phenotypic characteristics, suggesting an interrelationship of proteins in a complex or in a linear pathway. To date, 12 genes have been cloned, but the encoded proteins bear no resemblance to each other or to any other known proteins. Several studies have revealed a cell cycle specific phenotype in response to DNA damage, implying a cell cycle checkpoint role for putative FA proteins. My work has focused on protein-protein interactions and has characterized the binding of the G2/M cyclin dependent kinase, cdc2, to the first described FA protein, FANCC (Fanconi Anemia group C). Since the cloning of FANCA, I have raised an antibody against FANCA and described the binding of FANCA and FANCC in a nuclear complex. Now I have data showing that the FANCA-FANCC protein complex is sizable (2 MD) and have embarked on efforts to search for other protein partners to gain further clues on the function of the FA pathway. We also continue to assess the role of the FA proteins in cell cycle regulation and in the subnuclear environment.

Based on our interest on marrow failure and genomic instability, we have also started working on 3 related projects. First, we have begun to purify the protein complexes containing gene products that are defective in 2 additional hematopoietic failure syndromes, Diamond-Blackfan anemia (DBA) and congenital dyserythropoietic anemia (CDA). As in FA, the proteins (RPS19 for DBA, codanin for CDA) have no known function, and additional genes accounting for additional genetic complementation groups remain to be cloned and identified.

Second, we are investigating ways to use our knowledge of genomic instability for improving cancer therapeutics. We have been working on tax1, a viral oncogene, in collaboration with the Semmes laboratory at Eastern Virginia Medical School. Interestingly, tax1 chemosensitizes p53 mutant cells in culture. This observation is especially important, as p53 mutations are found in a majority of all human cancers and are the leading cause of resistance to chemotherapy. Our goal is adapt the tax1 effect on cells with p53 mutations in order to make cancer therapy more effective in resistant tumors.

Finally, we have also started a more clinical project, using mass spectroscopy technology we have used to find FA binding proteins. Again in collaboration with the Semmes laboratory, we have adapted the mass spec to analyze sera from patients with pediatric malignancies in order to identify unique protein markers of disease. These markers could then be used for diagnosis, prognosis, staging, and tracking of minimum residual disease in patients. In addition, our goal is to identify interesting proteins for further analysis in our laboratory.

Recent Papers from the Kupfer Laboratory:

• Qiao, et.al. Fanconi Anemia Proteins Localize to Chromatin and Nuclear Matrix in a DNA damage and Cell Cycle Regulated Manner. JBC, 2001.

  Microscopic analysis of the subcellular localization of the FA proteins revealed a strong nuclear appearance, except in nucleoli, as well as exclusion from the nucleus during mitosis. As a result of this finding, we formally demonstrated that FA proteins were tightly bound to chromatin and became more so after DNA damage. In addition, we noted that one of the FA proteins, FANCG, became phosphorylated at mitosis, which signaled the exit of the entire FA core complex from the nucleus. The idea that the FA complex associates intimately with chromatin is consistent with idea that the normal action of the FA pathway is to maintain genomic stability.

• Qiao, et.al. Phosphorylation of Fanconi Anemia Complementation Group G Protein at Serine 7 is Important for Function of the FA Pathway. JBC, 2004.

  Our previous paper demonstrated that FANCG was phosphorylated. Using mass spectroscopy, we isolated FANCG peptides and found a peptide containing phosphoserine 7. Mutational analysis revealed that a FANCG protein mutated at this serine failed to fully correct a mutant FA cell line. In order to analyze the phosphorylation event more fully, we made a phosphospecific antibody, which detected that FANCG became more phosphorylated after DNA damage and during the S phase of the cell cycle. We concluded that this phosphorylation event was functionally important for the FA pathway to act normally.

• Mi, et.al. FANCG is Phosphorylated at Serines 383 and 387 during Mitosis. MCB, 2004.

  We earlier noted that FANCG was phosphorylated specifically during mitosis. Again using mass spectroscopy, we narrowed the phosphorylated region to a small part of the protein. Two candidate sites were mutated (serine 383 and serine 387), and these mutations abrogated the ability of FANCG to correct mutant cells. Furthermore, we demonstrated that the FANCG protein interacted with the cdc2 kinase, which is responsible for propelling the cell into division (mitosis) after DNA replication. These data indicate that the interaction of the FA complex with genetic material is tightly regulated via phosphorylation events.

• Thomashevski, et.al. The Fanconi Anemia Core Complex Forms Four Complexes of Different Sizes in Different Subcellular Compartments. JBC, 2004.

  While 9 genes for FA have been cloned, the proteins resemble no know proteins and have no functional motifs. Therefore, it is not understood what the normal biochemical function of the FA pathway is. A long term goal of our lab is to purify other proteins in the FA protein complex that do have a known function, thus helping us understand the overall function of the FA pathway. Using a series of chromatographic techniques to fractionate protein, we separated and isolated the FA core complex into 4 distinct sizes, depending on the which part of the cell was analyzed. We have used mass spectroscopy to identify new proteins and are currently in the midst of analyzing several interesting binding proteins.

• Mi and Kupfer. The Fanconi Anemia Core Complex Associates with Chromatin during S Phase. Blood, 2005.

  Building on our earlier observation that the FA proteins bind to chromatin, we further investigated the stimuli for such an observation. We tagged FANCA, FANCC, and FANCG with fluorescent proteins and introduced them into cells. These cells were then synchronized into various parts of the cell cycle or treated with DNA damaging agents. The cells were then analyzed by microscopy. These experiments revealed a dynamic transport of the FA complex from cytoplasm to nucleus in time for DNA replication, as exemplified by real time motion pictures.

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