In the past funding period, the hypothesis that cells sense an oxidative event but signal through NO7 dependent protein post-translational mechanisms was tested. Two signal transduction mechanisms important in the cytoprotective response to ionizing radiation (IR) were examined: Tyr kinase signaling and activation of the transcription factor NF-?B. IR at clinically relevant doses (<5 Gy), activates Ca2+ dependent NOS, resulting in the transient S-nitrosation of PTP active site Cys and inhibition of cellular PTPs. One consequence in autocrine-regulated tumor cells is IR-enhanced growth factor receptor Tyr kinase signaling. These findings demonstrated the first mechanism that accounts for the common observation that IR and other mild oxidative stresses activate cytoprotective Tyr kinase signaling pathways. These same IR doses also stimulated NK-?B activity by a mechanism involving the transient nitration of the inhibitor protein, I?B?. The latter finding has prompted the present proposal to test the hypothesis that IR-induced Tyr nitration of key regulatory proteins is a specific post-translational modification responsive to mild oxidative/nitrosative stresses such as IR.
Specific Aim 1 is focused on Herein, the expression and activity levels of eNOS and iNOS in MCF-7 breast carcinoma cells and in tumor endothelial cells in vitro and in vivo are monitored as a function of dose (1-10 Gy) and time (up to 48 hrs post-IR). Immunocytochemical and subcellular fractionation methods identifies sites of nitration and NOS localization. The role of infiltrating inflammatory cells that express high levels of iNOS is examined by depleting tumors of the infiltrating cells with anti-GR-1.
Specific Aim 2 a global approach to identify by mass spectroscopy key regulatory proteins nitrated after IR and a physical chemical modeling and genetic validation study to identify structural motifs predictive of nitration.
Specific Aim 3 tests whether specific nitration of p53 has a role in the cellular response to radiation. Preliminary studies demonstrated that p53 is transiently nitrated after IR. Mass spectroscopy identified two nitratable tyrosine including Tyr327 in the tetramerization domain and Try107 in the DNA binding domain. Functional consequences of their nitration in terms of mitochondrial and nuclear localization, interaction with Bcl molecules, and transcriptional responses are examined. Demonstrating a functional consequence is critical in establishing the physiological significance of the post-translational modification. These studies will validate the role of Tyr nitration as a signal transduction mechanism responsive to oxidative/nitrosative stresses and may provide new strategies to enhance the therapeutic ratio in the treatment of cancer.

Public Health Relevance

This proposal evaluates a novel signaling mechanism involving nitric oxide and activated by radiation and other mild oxidative stresses. Understanding the functional consequences of this signaling mechanism is important in the development of new strategies to enhance the therapeutic efficacy of radiation. New mechanisms contributing to oxidative/nitrosative induced carcinogenesis may also be identified.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA090881-09
Application #
8268474
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Bernhard, Eric J
Project Start
2001-01-01
Project End
2013-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
9
Fiscal Year
2012
Total Cost
$266,245
Indirect Cost
$88,155
Name
Virginia Commonwealth University
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
Rabender, Christopher S; Alam, Asim; Sundaresan, Gobalakrishnan et al. (2015) The Role of Nitric Oxide Synthase Uncoupling in Tumor Progression. Mol Cancer Res 13:1034-43
Alam, Asim; Mukhopadhyay, Nitai D; Ning, Yi et al. (2015) A Preliminary Study on Racial Differences in HMOX1, NFE2L2, and TGFβ1 Gene Polymorphisms and Radiation-Induced Late Normal Tissue Toxicity. Int J Radiat Oncol Biol Phys 93:436-43
Yakovlev, Vasily A (2015) Role of nitric oxide in the radiation-induced bystander effect. Redox Biol 6:396-400
Yakovlev, Vasily A (2013) Nitric oxide-dependent downregulation of BRCA1 expression promotes genetic instability. Cancer Res 73:706-15
Cardnell, Robert J G; Rabender, Christopher S; Ross, Gracious R et al. (2013) Sepiapterin ameliorates chemically induced murine colitis and azoxymethane-induced colon cancer. J Pharmacol Exp Ther 347:117-25
Zhang, Qifang; Raje, Vidisha; Yakovlev, Vasily A et al. (2013) Mitochondrial localized Stat3 promotes breast cancer growth via phosphorylation of serine 727. J Biol Chem 288:31280-8
Guo, Chunqing; Yi, Huanfa; Yu, Xiaofei et al. (2012) In situ vaccination with CD204 gene-silenced dendritic cell, not unmodified dendritic cell, enhances radiation therapy of prostate cancer. Mol Cancer Ther 11:2331-41
Cardnell, Robert J G; Mikkelsen, Ross B (2011) Nitric oxide synthase inhibition enhances the antitumor effect of radiation in the treatment of squamous carcinoma xenografts. PLoS One 6:e20147
Bayden, Alexander S; Yakovlev, Vasily A; Graves, Paul R et al. (2011) Factors influencing protein tyrosine nitration--structure-based predictive models. Free Radic Biol Med 50:749-62
Yakovlev, Vasily A; Mikkelsen, Ross B (2010) Protein tyrosine nitration in cellular signal transduction pathways. J Recept Signal Transduct Res 30:420-9

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