The p38 MAP kinase (MAPK) pathway is activated in response to stress stimuli (e.g. ionizing irradiation) that induce DNA double stranded breaks (DSBs) and plays a role in the induction of cell cycle checkpoints (primarily G2/M) to facilitate DNA repair. DSBs are also generated during the development of T cells while undergoing V(D)J recombination in the thymus. V(D)J-mediated DSBs (coding and signal ends) trigger a DNA damage response and DNA repair similar to those induced by DSBs generated by ionizing irradiation. Although joining of coding ends is critical for the formation of functional TCR, repairing of signal ends flanking the excised DNA fragment is also important to prevent integration of the DNA fragments randomly in the genome and genomic instability. We have previously shown that p38 MAPK is activated in double negative (DN)3 thymocytes undergoing V(D)J recombination of the TCR2 and contributes to the induction of a G2/M cell cycle checkpoint. In addition, p38 MAPK also provides survival to DN3 thymocytes. Our recent studies show that p38 MAPK phosphorylates GSK32 at a non-previously characterized residue (Ser389) and this phosphorylation inactivates GSK32. Inactivation of GSK32 is associated with increased survival. We propose that p38 MAPK is activated in DN3 thymocytes by the DNA damage response (Atm activation) triggered by V(D)J-mediated DSBs and contributes to repairing of signal ends and prevention of signal end integration not only by inducing a cell cycle checkpoint, but also by promoting survival through inactivation of GSK32. We will investigate whether: 1) activation of p38 MAPK in DN3 thymocytes is mediated by ATM as a result of the DNA damage response triggered by V(D)J-mediated DSBs (Aim 1);2) p38 MAPK provides survival signals to DN3 thymocytes by phosphorylating and inactivating GSK32 (Aim 2);3) activation of p38 MAPK promotes the repair of signal ends and diminishes integration of signal end fragments in the genome (Aim 3). Public Health Relevance: DNA damage and genomic instability are clearly associated with the development of malignancies. To prevent genomic instability, cells normally undergo cell cycle checkpoints (G1/S and G2/M checkpoints) that delay the normal cycle to allow time for DNA repair to occur. Although UV and ionizing irradiation as well as chemotherapeutic drugs are the major inducers of DNA damage, genomic instability is continuously present in developing T and B cells while undergoing recombination of their corresponding T cell receptor (TCR) or BCR genes. During this process, denominated V(D)J recombination, fragments of DNA with non-protected double stranded breaks (signal ends) are excised from the genome. These signal end DNA fragments can be a hazard for T and B cells since they can attack other duplex DNA and can randomly integrate into the genome, causing potential malignancies. The frequency of T and B cell leukemias is relatively high and often these malignancies have been associated with chromosomal translocations, deletions and insertions that affect the expression or activity of specific proto-oncogenes. The establishment of checkpoints during the generation of T and B cell receptors is therefore important to maintain genomic stability. We propose to investigate the role that the p38 MAP kinase pathway has in preventing integration in the genome of signal ended fragments resulting from TCRb V(D)J recombination in immature thymocytes (DN3 thymocytes), by promoting the induction of a cell cycle checkpoint (primarily G2/M) and also providing survival. Pharmaceutical inhibitors of the p38 MAPK pathway are currently in clinical trials. If our hypothesis is correct, these inhibitors should be avoided primarily in infants where T cell development is highly active. In addition, genetic mutations that may affect this pathway could determine a relatively higher susceptibility to develop T cell malignancies.

Public Health Relevance

DNA damage and genomic instability are clearly associated with the development of malignancies. To prevent genomic instability, cells normally undergo cell cycle checkpoints (G1/S and G2/M checkpoints) that delay the normal cycle to allow time for DNA repair to occur. Although UV and ionizing irradiation as well as chemotherapeutic drugs are the major inducers of DNA damage, genomic instability is continuously present in developing T and B cells while undergoing recombination of their corresponding T cell receptor (TCR) or BCR genes. During this process, denominated V(D)J recombination, fragments of DNA with non-protected double stranded breaks (signal ends) are excised from the genome. These signal end DNA fragments can be a hazard for T and B cells since they can attack other duplex DNA and can randomly integrate into the genome, causing potential malignancies. The frequency of T and B cell leukemias is relatively high and often these malignancies have been associated with chromosomal translocations, deletions and insertions that affect the expression or activity of specific proto-oncogenes. The establishment of checkpoints during the generation of T and B cell receptors is therefore important to maintain genomic stability. We propose to investigate the role that the p38 MAP kinase pathway has in preventing integration in the genome of signal ended fragments resulting from TCRb V(D)J recombination in immature thymocytes (DN3 thymocytes), by promoting the induction of a cell cycle checkpoint (primarily G2/M) and also providing survival. Pharmaceutical inhibitors of the p38 MAPK pathway are currently in clinical trials. If our hypothesis is correct, these inhibitors should be avoided primarily in infants where T cell development is highly active. In addition, genetic mutations that may affect this pathway could determine a relatively higher susceptibility to develop T cell malignancies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI051454-10
Application #
8416381
Study Section
Cellular and Molecular Immunology - B Study Section (CMIB)
Program Officer
Prabhudas, Mercy R
Project Start
2002-06-01
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2015-01-31
Support Year
10
Fiscal Year
2013
Total Cost
$346,638
Indirect Cost
$116,314
Name
University of Vermont & St Agric College
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
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