Molecular radiobiology faculty
Ross B. Mikkelsen, Ph.D.
Aylin Rizki, Ph.D.
Kristoffer Valerie, Ph.D.
Vasily A. Yakovlev, MD, Ph.D.
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Dr. Mikkelsen initially demonstrated that ionizing radiation in the clinically relevant dose range stimulates significant transient changes in cytosolic [Ca2+] in all epithelial tumor cell lines examined. These studies have been extended to analyses of mechanisms by which cells sense radiation and metabolically produced reactive oxygen, and how resulting signals are amplified and translated into cellular responses. Dr. Mikkelsen’s laboratory demonstrated that one cytoplasmic mechanism involves a mitochondrial oxidative event that is propagated via a Ca2+ dependent reversible mitochondrial permeability transition. A consequence is the activation of constitutive, Ca2+ dependent nitric oxide synthases. The generation of NO provides a mechanism by which cells can modulate signal transduction pathways including MAPK and cGMP-dependent kinases and the redox sensitivities of different transcription factors. NO also out competes superoxide dismutase for superoxide and thus the low level NO generation by constitutive NO synthases provides a mechanism for buffering localized increases in cytotoxic, mutagenic superoxide/hydrogen peroxide. Because NO is relatively stable and lipophilic, NO generation potentially provides a mechanism by which an oxidative event in one cell can be signaled to adjacent cells. Current studies are examining Cys S-nitrosylation and Tyr nitration as protein modification mechanisms by which NO activates cellular responses to oxidative events and the role of NO synthases in reducing oxidative base damage as a consequence of radiation exposure and metabolic activity. These studies are using traditional genetic approaches combined with mass spectroscopy to identify the modified amino acids and the functional consequences of modification. Dr. Mikkelsen’s laboratory also collaborates with Dr. Glen Kellogg of Medicinal Chemistry and Structural Biology in analysis of protein structural features that are determinants of Tyr nitration following an oxidative/nitrosative stress. Recent studies in the lab demonstrated that radiation (or treatment of cells with low H2O2 concentrations) stimulated the reversible S-nitrosylation of the active site Cys of protein tyrosine phosphatases (SHP-1 and SHP-2) transiently inactivating the phosphatases. At least with respect to SHP-2, this oxidative modification provides a mechanism by which radiation activates the ERBB1 receptor. These studies are transitioning into animal studies with the focus on radiation-stimulated inflammatory responses. Dr Mikkelsen’s laboratory also investigates growth factor signaling and cellular response to radiation with specific emphasis on how the early reactive oxygen/nitrogen signal activates growth factor receptor regulated pro-proliferative and anti-apoptotic signaling. The important findings from these studies are: (1) the identification of ERBB molecules as key mediators of cellular responses, e.g. anti-apoptotic mechanisms, to radiation and other oxidative/nitrosative stresses; (2) the demonstration that oxidative activation of ERBB1 is different than ligand activation in terms of the mechanism consequences; and (3) the mechanism and consequences of radiation-induced nuclear import of ERBB1. Recent Publications: Barrett, DM, Black SM, Todor H, Schmidt-Ullrich RK, and Mikkelsen, RB. Inhibition of Protein Tyrosine Phosphatases by Mild Oxidative Stresses is Dependent on S-Nitrosylation, J. Biol. Chem. 280, 14453-14461, 2005; Sturla L-M, Amorino GP, Alexander MS, Mikkelsen RB, Valerie K and Schmidt-Ullrich RK. Requirement of Tyr992 and Tyr1173 in phosphorylation of the epidermal growth factor receptor by ionizing radiation and modulation by SHP2, J. Biol. Chem. 280, 14597-14601, 2005; Mikkelsen RB and Wardman P, Biological chemistry of reactive oxygen and nitrogen and ionizing radiation-induced signal transduction mechanisms, Oncogene Reviews, 22, 5734-5754, 2003. |
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The focus of the laboratory is the reciprocal interactions between extracellular matrix (ECM) and genome stability, as these relate to breast cancer progression and radiation therapy. Dr. Rizki’s laboratory aims to: 1-) Determine the mechanisms by which ECM signaling regulates DNA double-strand break repair. 2-) Determine the mechanisms of genome stability gene function in acquisition of invasiveness through ECM. To determine the mechanisms by which ECM signaling regulates DNA double-strand break repair, three research areas are established: CYTOPLASMIC SIGNALING COMPONENTS downstream of beta1 integrin that are important in regulation of double-strand break repair, NUCLEAR MECHANISMS targeted by the beta1 integrin mediated signaling events to regulate double-strand break repair, and RELEVANCE TO THE MAMMARY GLAND IN VIVO using mouse models. To determine the mechanisms of genes involved in Genome InStability and ECM Invasion or GISEM gene function in pre-invasive to invasive transition, three research areas are in progress: INVASION-SPECIFIC TARGETING by separating the invasion functions from genome stability using a mutagenesis-based approach, GISEM GENE IDENTIFICATION and mechanism determination, GISEM GENE SUBSET identification as potential markers of pre-invasive to invasive transition in breast cancer. SIGNIFICANCE. In general, understanding how the microenvironment and the processes important for genome stability interact is important for deciphering the mechanisms of tumor progression, discovering new types of drug targets in the treatment of cancer, and finding risk markers for progression and response to radiation therapy. |
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Dr. Valerie received his doctoral degree in Biochemistry in 1986 from the Royal Institute of Technology in Stockholm, Sweden. His thesis studies focused on the cloning and characterization of a small DNA repair gene from bacteriophage T4. He then went on to do post-doctoral training with Dr. Martin Rosenberg at Smith Kline and French Laboratories studying transcriptional regulation of the human immunodeficiency virus and stress-activated gene expression. He joined the Department of Radiation Oncology at VCU in 1989, and is now a Professor and Chair of the Division of Molecular Radiobiology. Research in our laboratory focuses on DNA double-strand break (DSB) repair, radiation-induced signaling and developing novel approaches for sensitizing tumor cells to radiation. A major thrust is on investigating the role of ataxia telangiectasia mutated (ATM) in regulating DSB repair. ATM is a member of the phosphatidylinositol-3’-kinase-like kinase (PIKK) family of serine-threonine protein kinases. The PIKKs are important for regulating cellular homeostasis and the coordination of cell growth, senescence, and DNA damage responses. ATM is critical for controlling the G1/S, intra-S, and G2/M checkpoints and is also believed to play an important role in the repair of certain hard-to-repair radiation-induced DNA damage lesions. We recently demonstrated that ATM is important for efficient homologous recombination repair (HRR) in human cells. HRR is an essential error-free but minor type of DSB repair whereas non-homologous end-joining (NHEJ) is the predominant type of DSB repair in human cells. NHEJ is considered more error-prone than HRR. More recent findings from our laboratory have shown that all three of the major signaling pathways, ERK, JNK and p38, modulate HRR. In particular, our work has demonstrated that the ERK pathway associated with pro-survival and cellular growth forms a regulatory feedback loop with ATM. Specifically, MEK/ERK signaling appears to regulate the ATM kinase activity and vice versa, suggesting that there is close coordination between growth, DNA repair and survival of irradiated cells. These relationships are currently being investigated in more detail. Another more recent avenue of investigation focuses on the role of BRCA1 in DSB repair. The tumor suppressor BRCA1 is associated with both HRR and NHEJ. BRCA1 appears to play a role in modulating the fidelity of DSB repair. We are currently investigating the role of phosphorylation of BRCA1 in DSB repair using DSB repair assays based on the I-SceI restriction enzyme system. We have developed technology that replaces wild-type BRCA1 with mutant forms by using inducible RNAi approaches. Preliminary results suggest that one of the major ATM BRCA1 phosphorylation sites, ie serine-1387, appears to regulate cytoplasmic-nuclear localization of BRCA1 that affects radioresistance and HRR. The effect of BRCA1 phosphorylation and other post-translational modifications on high-fidelity NHEJ are also being investigated. These studies are extended into identifying novel BRCA1-interacting proteins by mass spectrometry and radiation-induced post-translational modifications of BRCA1. We have constructed BRCA1 mutants that fail to bind phospho-proteins to the carboxy-terminal BRCT domains. We expect these mutants to be impaired in binding DNA repair accessory protein important for DSB repair. Many of our DSB repair studies focus on mechanisms occurring in brain cancer. Recent studies from other laboratories suggest that glioma stem cells have potent DNA damage checkpoints and DSB repair accounting for the unusual radioresistance of GBM. The long-term goal of our laboratory is to identify novel molecular targets in the DSB repair/signaling circuitry that could be used in combination with radiation to improve cancer therapy. |
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Dr. Yakovlev completed his medical education (M.D.) at the Russian State Medical University, Medical Faculty, Moscow, Russia in 1997. He did his Post-graduate Training in the Department of Combined Methods of Treatment and Laboratory of Experimental Diagnostic and Biotherapy of Tumors in the All-Russian Cancer Research Center, Moscow, Russia. His thesis work was done on molecular tumor markers as the factors of prognosis in the treatment of colorectal cancer. He joined the Department of Radiation Oncology at Virginia Commonwealth University in 2003 and did his post-doctoral training with Dr. Mikkelsen. During this training, Dr. Yakovlev’s research focused on the transcription factor, NF-kappaB, known to have cytoprotective roles in most tumors. NF-kappaB has also been previously shown to be activated by ionizing radiation and the inhibition of NF-kappaB sensitizes tumors to radiation and chemotherapy. Dr. Yakovlev’s work showed that activation of NF-kappaB at therapeutic doses of radiation differs from that at higher doses and involves tyrosine nitration of IkappaB protein (inhibitor of NF-kappaB). leading to dissociation of intact IkappaB from NF-kappaB. This mechanism does not appear to require IkappaB kinase dependent phosphorylation or proteolytic degradation of IkappaB. The mechanism he has described not only explains the radiation effect but also may explain why NF-kappaB activity is higher in tumors than normal tissues. This work also connects the inflammatory state that generates high levels of nitric oxide (NO) and tumor cell resistance. In addition, using a drug that blocks the generation of NO, which is currently used in clinical trials, it was shown, in unpublished studies, that inclusion of this relatively non-toxic drug in the drinking water of mice significantly reduces tumor growth. Recently, Dr. Yakovlev extended these studies to another critical player of carcinogenesis – p53 protein. High doses of NO can activate p53 by inducing DNA damage and ATM/ATR-dependent p53 phosphorylation. Dr. Yakovlev has demonstrated that low levels of NO promotes p53 tetramerization and activation by completely different mechanism: Tyrosine 327 nitration (publication under preparation). Activation of p53 by low doses of NO, however, is ATM/ATR-independent and does not lead to p53 phosphorylation. Moreover, different mechanisms of p53 activation by low- and high-doses of NO result in different patterns of p53-target genes modulation. These studies have directed my research into defining the role of increased level of NO in the regulation of genetic and chromosomal instability during chronic inflammation. Clinical studies have documented an association between inflammation and carcinogenesis. Inflammatory cells, such as neutrophils, macrophages, and eosinophils, are an important endogenous source of ROS/RNS. Whereas most investigations have focused on genotoxic effects, the impact of ROS/RNS in regulation of mechanisms of genetic and chromosomal stability is poorly understood. |
