DNA double stranded breakes(DSB) physiologically occur at various stages during B lymphocyte development at the Immunoglobulin(IG) chromosomal loci.First, DSB are caused by V(D)J recombination which reshuffles the germline gene segments to assemble functional genes encoding the variable regions of their Ig receptors.In the bone marrow, recombination occurs first during Pro-B cell stage at their heavy chain locus IgH(chromosome 14 in humans-chr.14) and later either at their light IgL-k locus(chr.2) or chr.22) during Pre-B stage.Once an Ig B cell receptor(BCR) is expressed on the cell surface the immature naive B cells become circulant and get recruited in the spleen where they await antigen and lymphocyte TH cell encounter to progress to mature B and later to plasma cells.During these later stages a third round of physiologic DSBs occur and are associated with somatic hypermutation(SHM-antibody affinity maturation) and isotype switching(from IgM to IgG) processes, together termed BCR stimulation.When DSB at IG loci are coupled with other accidental DNA lesions somewhere else in the genome the concurrent set of events lead to reciprocal translocations, which are exchanges of encoding DNA segments between two distinct non-homologous chromosomes.Translocations can either change the integrity of some incriminated encoded protein factors generating chimaeric or modified polypeptides, or the levels of their expression profile (when the regulatory regions of their genes get modified).Although for B cells these translocations are recognized hallmarks of their malignant transformation it is unknown if they are the cause of, or if their presence just favors lymphomagenesis(the process of oncogenic degeneration of the lymphoid cell lines).Our project addresses this essential question trying to capture in time the exact moment when a given induced translocation(MYC/IG) abruptly changes the phenotype of B cells undergoing differenti-ation.We will study the effects of induced translocations between an IG and MYC locus(chr.8) occurred at the above mentioned stages in B cell development and then from the analysis of the genomic mapping we will establish correlations between the severity of the cellular phenotypic changes and the type of genomic DNA lesion incurred.Our main goal is to establish a direct correlation between these physiologic events that cause DSBs during B cell maturation and MYC locus translocations involved in the lymphomagenesis of some of the most fearsome cancers of lymphoid tissues shown to be resilient to modern and conventional therapy.c-MYC is a transcription factor that orchestrates the levels of expression of many of the genes involved in a wide variety of cellular activities(cyclins, ribosomal proteins or DNA replication machinery factors) from very early embryonic to fully differentiated stages of development.Above all, c-MYC directs many cell cycle regulation genes crucially involved during all stages of B cell development(bcl2, bcl6, TCF3, NFkB).As a consequence, IG/MYC translocations are frequently found and are used as associated biomarkers for the diagnostic of most fearsome B cell lymphomas with negative prognostic and poor treatment response. In this category one includes Acute B Lymphoblastic Leukemia(B-ALL), nonHodgkin B cell lymphomas(NHLB) like Burkitt lymphoma(BL) or Diffuse large B cell lymphomas(DLBCL), cancers which will provide the phenotypic guiding markers to orient our study.The underlying idea of our proposal is to couple the physiologic DSBs(occurred in pre-B cells undergoing V(D)J recombination or in immature B cells at BCR stimulation), with  induced irradiation DSBs occurred at MYC locus to favor the incidence of abnormally DNA repaired translocations.To target the effects of irradiation towards c-myc gene the cells will be specifically stimulated with GMCSF, a growing factor that enhances both the transcription level and chromatin decondensation at MYC locus.Therefore the c-myc gene DNA becomes more susceptible to the torsional stress of sustained RNA pol II action which at the same time unshields it from the nucleosomal protection.Both these effects will expose c-myc gene DNA making it more vulnerable to irradiation induced lesions. First, we need to understand how irradiation a condition that induces DSBs and cellular DNA damage response affects the growth and development of cultures of murine pre-B cells (vAbl transformed pre-B), and naive B cell lines(human and murine), maintained under various stimulatory conditions.We will correlate the irradiation dose exposure with the extent and locations of DNA lesions which are compatible with cell survival.We will assess the induced DNA fragility at the MYC locus under steady state or transcription stimulation and how such effects are reflected in the expression of stage specific surface B cell markers.At cellular level we will correlate the number and location of DNA repair foci(H2AX) with the irradiation dose.We will target oncogene translocations during V(D)J recombination and BCR stimulation.Pre-B/B cells will be separately or cumulatively subjected to a combination of four types of actions:irradiation,V(D)J recombination,BCR signaling, and individual stimulation/inhibition of transcription of c-myc oncogene.Following these simple or combined exposures the genomic DNA from these cells will be specifically investigated for translocations between IG loci and the MYC locus.We should establish a representative number of degenerated B cell clones with detected translocations.We will correlate the MYC-IG translocations structure with the expression of various internal and surface B cell markers.The central idea of the proposal is to find how the anatomy of various MYC-IG loci fusions are reflected in the expression profiles of the newly transformed malignant B cells.For this we will sequence all MYC-IG translocations found in our B cell clones and analyze their protein expression profiles versus the background of normal B cells.We will show how the differential protein expression profiles affect the dynamics of BCR,CD10,CD19 and CD20 surface markers.We will establish correlative indices between each type of translocation topology and transformed B cells resembling phenotypically the prototype guiding lymphoma cells found in patients.