BACKGROUND
double strand  dna breaks are generated in a variety of ways from both genotoxic and physiologic sources.
in developing lymphocytes one source of physiological double strand breaks  is the process of vj recombination that is utilized to generate rearranged antigen receptor genes  <cit> .
this process is initiated by rag endonucleases while the lymphocytes are in the g <dig> phase of the cell cycle.
these breaks are necessary to create the vast diversity seen in lymphocyte antigen receptors.
in addition to physiologic breaks, lymphocytes are exposed to a variety of genotoxic damage from exogenous sources.
one such damage source is ionizing radiation , which can be generated from both natural and man-made sources including radon gas and medical devices and procedures.
ionizing radiation can cause dsbs, as well as other dna lesions, and has been shown to disrupt many cellular functions.
failure to properly repair this damage can lead to detrimental health effects, such as uncontrolled cell death and cancer formation.
the normal response to dsdna breaks includes the activation of multiple transcriptional pathways that can lead to cell cycle arrest at specific checkpoints, dna repair, or death of the affected cells  <cit> .
these genome wide transcriptional responses are very tightly regulated and complex.
they also differ between different cell types  <cit> , possibly depending on different sensitivities to dna damage in general and to different cellular functionalities.
here we compare the response of developing b cells to both physiologic and genotoxic dsbs.
in a previous study we showed that physiological dna dsbs induced in the g <dig> phase of the cell cycle by the rag endonuclease-associated process of vj recombination activated a broad transcriptional profile with many regulated genes involved in diverse processes important for lymphocyte development  <cit> .
while it is known that genotoxic agents, such as ir, activate transcriptional programs involved in maintaining the integrity of the genome, we also want to investigate whether or not the genotoxic breaks could affect lymphocyte-specific maturation transcriptional responses similar to those we observed following rag-induced physiological dsbs.
by comparing the transcriptional responses to both types of dna damage, we can compare the similarities in the responses to damage as well as the differences induced by genotoxic damage.
similarities in the responses could indicate that genotoxic dna breaks are potentially disrupting normal cellular functions that occur in developing b cells, thus corrupting these developmental processes.
elucidation of the similarities and differences in these responses may lead to a greater understanding of the cellular mechanisms involved in lympho-proliferative cancer formation and lymphocyte maturation.
in this study we highlight that genotoxic dna damage not only activates a lymphocyte-specific transcriptional response but also activates a potentially hazardous transcriptional profile that includes a set of genes and pathways indicative of a cancer-predisposition.
RESULTS
ionizing radiation induces a broad transcriptional program in wild type murine pre-b cells
utilizing affymetrix whole mouse genome gene expression microarrays, we collected gene expression data from viral -abl kinase-transformed pre-b cell lines that were generated from multiple wt mice expressing an eμ-bcl <dig> transgene.
treatment of these pre-b cell lines with the abl kinase inhibitor, sti- <dig>  leads to a block in the g1-to-s cell cycle transition  <cit> .
cells blocked at the g1-to-s transition with sti- <dig> were evaluated for their gene expression response to mock treatment or exposure to 1 gy ionizing radiation .
the purpose of arresting these cells is to ensure that the cells are in the same phase of the cell cycle as the rag-induced physiologic response to ensure that similar dna damage repair processes would be available under both ir and physiologic conditions.
we recognize that this will negate detection of some of the gene expression patterns normally identified in logarithmically growing cells that are associated with cell cycle arrest and dna damage response changes following exposure to ir.
in our previous study we provided evidence that the rag dsb gene expression changes observed in the abl kinase-transformed cell lines was very similar to that of primary cells.
we expect the same to be the case for the ir response.
in order to identify significant changes in gene expression after ir-induced dna damage, we utilized a combination of t-test generated p-values and fold change cut-offs  ≥ ±  <dig> ) based on global gene expression analysis of  <dig> wt pre-b cell lines exposed to  <dig> or 1 gy ir.
we identified  <dig> probes that were significantly changed after ir exposure .
using ingenuity pathway analysis   <cit>  and gene set enrichment analysis   <cit> , we identified a broad range of pathways and gene families with significant responses to induction of genotoxic dna damage.
figure 1a shows a list of some of the top ipa canonical pathways significantly affected by exposure to ir.
the identification of gene families, from the gsea molecular signatures database, gives a functional overview of the genes identified as significantly altered after ir.
gene families share common features such as biochemical activity and homology.
gene family analysis shows  <dig> transcription factors as well as  <dig> protein kinases whose expression levels are significantly altered after ir.
this reflects the broad nature of the transcription program initiated by genotoxic dna damage .
physiologic and genotoxic damage induce a shared lymphocyte-specific response
in previous work  <cit> , we identified a lymphocyte-specific pattern of gene expression in response to physiologic dsdna breaks induced by rag endonuclease cleavage in primary pre-b cells and abl pre-b cell lines.
here we examined the similarities in gene expression changes in response to both physiologic  and genotoxic damage.
we identified  <dig> probes representing genes that are significantly regulated in the same direction in response to both damage types .
since our wt cells exposed to ir are wt for rag endonuclease, there may be a contribution of naturally occurring rag-induced breaks in addition to the ir-induced dna damage in these cells and the gene expression response may contain elements of responses to both types of dna lesions.
however, we expect the contribution of the rag-induced breaks to be minor since both the control and irradiated cells should respond in a similar manner to the sti- <dig>  and since these cells are also wt for the repair pathway machinery that rapidly resolves these breaks.
the similarly regulated genes include many lymphocyte specific maturation genes such as cd <dig>  cd <dig>  swap <dig>  and nfκb .
while these genes are regulated in response to both damage types, one difference we observed was that genotoxic damage appears to induce a more robust change in many of the affected genes and pathways.
ipa reveals affected canonical pathways consistent with b cell maturation .
since cd <dig> expression plays an important role in b cell maturation and its mrna levels are increased after dna damage, we compared the protein expression of cd <dig> after physiologic and genotoxic damage by flow cytometric analysis.
we observed the expected increase in the number of cells with increased cd <dig> surface expression 90 minutes after 1 gy ir  and this is in agreement with our previously published data showing an increase in cell numbers with higher cd <dig> expression levels after rag-induced dna breaks  <cit> .
genotoxic damage, but not physiologic damage, induces a potential cancer susceptibility cellular response
in addition to the similarities in response to both types of dna damage, we observed a robust gene expression profile after genotoxic damage that was not seen after the physiologic damage.
we identified  <dig> probes, representing almost  <dig> unique genes, which were differentially regulated in the response to ir but not in the response to physiologically induced damage .
the transcriptional response unique to the ir-induced damage includes increased expression of  <dig> oncogenes,  <dig> protein kinases and  <dig> transcription factors, as well as decreased expression of  <dig> tumor suppressors and  <dig> transcription factors.
changes in the expression in these broad-range signalling molecules suggest a diverse biological response to genotoxic dsbs.
in order to understand the broad biological mechanism and pathways affected by ir-induced dsbs, we utilized ipa to investigate the changes in pathways and biological functions caused by genotoxic damage .
several canonical pathways were affected in response to ir that were not seen to be affected in physiologic-induced profiles, such as an nrf2-mediated oxidative stress response and cell cycle regulation pathways.
as mentioned above, we also see a stronger enrichment of pathways associated with activation of mature b cells in response to antigen.
initial inspection of the genes significantly regulated by ir-induced damage revealed several oncogenes and tumor suppressor genes whose expression change correlates with changes reported to be involved in cancer formation.
these included increased expression of known proto-oncogenes, such as kras <cit>  and rras <cit> , and the oncomir microrna- <dig>  <cit> , as well as suppression of the expression of socs <dig>  a known tumor suppressor  <cit>  .
while the affymetrix mouse genome  <dig>  genechip array is not specifically designed to recognize micrornas, the current annotation of the array revealed that several micrornas are represented in the array.
microrna- <dig> is known to target socs <dig> and this combination of increased expression of mir- <dig> and suppression of socs <dig> has been described in several b cell-derived lymphomas  <cit> .
our analysis shows many biological functions affected that are consistent with over-expression of potential oncogenes, such as cancer formation, cellular proliferation, and cell-mediated immune responses.
increased mir- <dig> expression suggests an increased capacity for cellular proliferation after ionizing radiation
increased expression of mmu-mir- <dig> in response to ir-induced damage suggests an increase in the capacity for proliferation  <cit>  of developing b cells when exposed to genotoxic stress.
mature and functional micrornas are processed through a series of steps from a primary microrna transcript.
it is possible the change in the mmu-mir- <dig> levels seen on the microarray is a reflection in the change of the primary mir- <dig> transcript.
in order to investigate the changes of the mature microrna levels after ir-induced dsbs and to further examine investigate the expression of mir- <dig> and its target socs <dig>  we undertook a time course to analyze and validate the mir- <dig> response to genotoxic damage over time.
we used quantitative real-time pcr  to track the mir- <dig> and socs <dig> expression levels at  <dig>   <dig>  and 8 hours post ionizing radiation in the  <dig> wt lines.
a robust increase of mir- <dig> expression levels is seen in the initial hours after genotoxic damage, with an average of  <dig>  and  <dig>  fold increases seen at  <dig> and 4 hours, respectively, post ir.
by 8 hours post ir it appears the levels of mir- <dig> are returning to baseline.
in an inverse correlation, socs <dig> expression is robustly decreased initially after ir exposure and has almost returned to baseline levels by 8 hours post damage  and additional file  <dig> ).
activation of nrf <dig> after ir-induced dna damage suggests a cellular protective response to oxidative stress
one pathway we observed to be regulated after genotoxic but not physiologic damage was the nrf <dig> oxidative stress pathway.
members of the nrf <dig> signalling pathway, including maff, sqstm <dig>  and txnrd <dig>  were up regulated after exposure to genotoxic dna damage by microarray.
since ir exposure is known to induce oxidative stress  <cit> , induction of this pathway was expected, but in combination with the potential for increased proliferation suggested by mir- <dig> expression, it may reflect a potentially dangerous, cancer predisposed situation in response to genotoxic damage.
in order to investigate and validate the induction of an nrf <dig> response to ir, we utilized a time course post ir to examine investigate nrf <dig> protein expression levels as well as mrna expression levels of the downstream target txnrd <dig> in each of the  <dig> wt pre-b lines .
changes in nrf <dig> protein expression were determined by quantitation from western blots of the  <dig> wt lines, with each nrf <dig> band adjusted for the loading control β-actin.
once activated, nrf <dig> protein accumulates in the nucleus, where it transactivates numerous target genes  <cit> .
in response to ir exposure, nrf <dig> protein levels were found to be on average  <dig> fold higher than in mock-irradiated controls at 2 hours post treatment.
levels continue to increase, reaching a peak at 4 hours post ir exposure.
levels of nrf <dig> are almost completely returned to baseline levels after 8 hours.
one downstream target of nrf <dig> is thioredoxin reductase  <dig> , which plays a role in protecting cells from oxidative stress  <cit> .
as expected, mrna levels of txnrd <dig>  as measured by qrt-pcr and normalizing to 18s levels, are increased at both  <dig> and 4 hours post genotoxic damage.
by 8 hours the levels of txnrd <dig> are returning to normal levels .
discussion
in this study we evaluated the transcriptional response induced by physiologic and genotoxic dsbs in developing b cells.
by comparing these different types of damage we found that there are important similarities as well as striking differences in the cellular responses to these different forms of dna lesions.
in previous work  <cit> , we observed a lymphocyte-specific response to physiologically generated rag-induced dsdna breaks.
highlights of this response include changes in the expression of genes important in immune function and maturation.
changes in these genes suggest an increase in the signalling of the cd <dig> and nfκb pathways, suggesting a role for dna breaks in the progression of b cell maturation.
after the observation that rag-induced dsbs trigger a response to move the b cells toward maturation, the next obvious question is whether or not other types of dsbs induce the same lymphocyte-specific maturation profile.
here we observed  <dig> probes that were differentially regulated in the same direction after induction of breaks regardless of the source of the damage.
these changes include increased expression of cd <dig>  cd <dig>  icam <dig>  swap <dig>  nfκb, as well as other immune related genes.
increased expression of these genes and others associated with cd40-, cd69-, and nfκb-related pathways suggest that the b cells are preparing to undergo maturation irrespective of the source of the dna damage.
while a core set of genes is regulated in the same direction after both types of damage, the response to genotoxic damage is generally more robust than the response to rag-induced breaks.
we hypothesize this is due to the greater amount of dna damage induced by ir exposures, perhaps initiating a stronger signal towards maturation.
while we see similarities in the response to physiologic and genotoxic breaks, we recognize there are potential biological and technical differences in comparing rag-induced and ionization radiation-induced dsbs.
rag-induced dsbs are tightly regulated and only a small number of breaks in very specific locations in the genome are induced.
in contrast, ir exposure can induce a broad range of dna damage, including dsbs, that is not restricted to specific locations but occurs throughout the genome and can involve dna lesions with damaged nucleotide ends as opposed to the “clean” ends generated by the rag endonucleases.
we have attempted to mitigate these differences by using exposures to a low dosage of ir and by ensuring the cells are in the same phase of the cell cycle to ensure that similar dna damage repair processes would be available under both conditions.
despite the differences in the nature of the dsbs it appears that the cells undergo a similar response to that damage by initiating a central lymphocyte-specific transcriptional response that is common to both.
in addition to these similarities, the genotoxic damage also induces changes in  <dig> unique probes representing almost  <dig> genes.
broad expression changes in transcription factors and protein kinases suggest genotoxic dsbs induce a myriad of changes in both gene expression and physiological pathways.
gene expression changes and alterations in pathways associated with b cell activation, increased proliferation, and oxidative stress responses are seen in the unique response to ir.
many of the pathways altered on a transcriptional level are known to be involved in the generation of cancers.
also, this study highlights the importance of additional layers of regulation, which has become obvious with the discovery of genotoxic regulation of small regulatory rnas such as micrornas .
their role in a wide variety of physiological processes has revealed their vital importance in proper cellular function, and disregulation has been linked to human diseases, including cancer and immune disorders .
in addition to the lymphocyte specific transcriptional pattern induced by both types of dna damage, genotoxic damage induces a potentially oncogenic combination of alterations of genes and biological response pathways.
we found that genotoxic, but not physiologic, damage induces increased expression of several proto-oncogenes such as kras and the oncomir mir- <dig>  this suggested to us that the cellular response to double strand dna damage could be specific to the method of generation of that damage, recognizing that ir-induced genotoxic damage causes many types of dna damage.
mir- <dig> is a known oncogenic mirna and its increase has been correlated with formation of b cell malignancies.
the up-regulation of mir- <dig>  at both the primary transcript and mature microrna level, seen after ir-induced breaks suggests a potential for the development of cancer after genotoxic damage.
additionally, mir- <dig> has been identified to suppress a number of tumor suppressors, including socs <dig>  which we found to be suppressed after genotoxic damage.
mir- <dig> up-regulation has been associated with b cell cancers and b cell transformation  <cit> , as well as with normal immune response  <cit> .
altered expression of this mirna and its target socs <dig> suggests that an increase in proliferation may be triggered after ir exposure.
interestingly, this increase in proliferation and up-regulation of mir- <dig> has been seen in mature b cells as a result of their response to antigen  <cit> .
another noteworthy difference between genotoxic and physiologic damage is the significant change in regulation of genes in b cell activation pathways and nrf2-mediated signalling.
nrf <dig> signalling is a known response to ir but has also been seen in the activation of mature b cells  <cit> .
the nrf <dig> pathway is a critical regulator of the defense against oxidative stress.
activation of nrf <dig> pathways is an important component in the clearing of oxidative stress and in a cytoprotective outcome.
there has been some suggestion that nrf <dig> also has a role in rescuing cells from cell cycle arrest that can be generated in response to oxidative damage  <cit> .
these responses to genotoxic damage in both the b cell activation and nrf <dig> signalling pathways could combine to result in serious deleterious consequences to the immune system.
CONCLUSIONS
the broad gene expression alterations, increased expression of the oncomir mir- <dig> and proto-oncogenes such as kras, the activation of nrf <dig>  and the lymphocyte specific maturation profile induced by genotoxic dsbs, reflect a potentially dangerous combination of conflicting signals for increased cellular proliferation and cytoprotective responses.
these conflicting signals could drive developing b cells to continue to mature and proliferate in the presence of dna damage after genotoxic exposures.
the possibility of maturation and proliferation in the presence of dna damage increases the risk for aberrant repair or lack of repair of damaged dna.
continued maturation and proliferation of these highly proliferative cells signalled by genotoxic dsbs provides a mechanism for the development of immunodeficiencies due to the potential loss of mature functional b cells as well as for the formation of lympho-proliferative cancers when dna repair is not completed successfully.

 double strand dna breaks are generated in a variety of ways from both genotoxic and physiologic sources. in developing lymphocytes one source of physiological double strand breaks is the process of vj recombination that is utilized to generate rearranged antigen receptor genes. this process is initiated by rag endonucleases while the lymphocytes are in the g phase of the cell cycle. these breaks are necessary to create the vast diversity seen in lymphocyte antigen receptors. in addition to physiologic breaks, lymphocytes are exposed to a variety of genotoxic damage from exogenous sources. one such damage source is ionizing radiation, which can be generated from both natural and man-made sources including radon gas and medical devices and procedures. ionizing radiation can cause dsbs, as well as other dna lesions, and has been shown to disrupt many cellular functions. failure to properly repair this damage can lead to detrimental health effects, such as uncontrolled cell death and cancer formation. the normal response to dsdna breaks includes the activation of multiple transcriptional pathways that can lead to cell cycle arrest at specific checkpoints, dna repair, or death of the affected cells. these genome wide transcriptional responses are very tightly regulated and complex. they also differ between different cell types, possibly depending on different sensitivities to dna damage in general and to different cellular functionalities. here we compare the response of developing b cells to both physiologic and genotoxic dsbs. in a previous study we showed that physiological dna dsbs induced in the g phase of the cell cycle by the rag endonuclease-associated process of vj recombination activated a broad transcriptional profile with many regulated genes involved in diverse processes important for lymphocyte development. while it is known that genotoxic agents, such as ir, activate transcriptional programs involved in maintaining the integrity of the genome, we also want to investigate whether or not the genotoxic breaks could affect lymphocyte-specific maturation transcriptional responses similar to those we observed following rag-induced physiological dsbs. by comparing the transcriptional responses to both types of dna damage, we can compare the similarities in the responses to damage as well as the differences induced by genotoxic damage. similarities in the responses could indicate that genotoxic dna breaks are potentially disrupting normal cellular functions that occur in developing b cells, thus corrupting these developmental processes. elucidation of the similarities and differences in these responses may lead to a greater understanding of the cellular mechanisms involved in lympho-proliferative cancer formation and lymphocyte maturation. in this study we highlight that genotoxic dna damage not only activates a lymphocyte-specific transcriptional response but also activates a potentially hazardous transcriptional profile that includes a set of genes and pathways indicative of a cancer-predisposition.  ionizing radiation induces a broad transcriptional program in wild type murine pre-b cells utilizing affymetrix whole mouse genome gene expression microarrays, we collected gene expression data from viral -abl kinase-transformed pre-b cell lines that were generated from multiple wt mice expressing an eμ-bcl transgene. treatment of these pre-b cell lines with the abl kinase inhibitor, sti- leads to a block in the g1-to-s cell cycle transition. cells blocked at the g1-to-s transition with sti- were evaluated for their gene expression response to mock treatment or exposure to 1 gy ionizing radiation. the purpose of arresting these cells is to ensure that the cells are in the same phase of the cell cycle as the rag-induced physiologic response to ensure that similar dna damage repair processes would be available under both ir and physiologic conditions. we recognize that this will negate detection of some of the gene expression patterns normally identified in logarithmically growing cells that are associated with cell cycle arrest and dna damage response changes following exposure to ir. in our previous study we provided evidence that the rag dsb gene expression changes observed in the abl kinase-transformed cell lines was very similar to that of primary cells. we expect the same to be the case for the ir response. in order to identify significant changes in gene expression after ir-induced dna damage, we utilized a combination of t-test generated p-values and fold change cut-offs ≥ ± ) based on global gene expression analysis of wt pre-b cell lines exposed to or 1 gy ir. we identified probes that were significantly changed after ir exposure. using ingenuity pathway analysis and gene set enrichment analysis, we identified a broad range of pathways and gene families with significant responses to induction of genotoxic dna damage. figure 1a shows a list of some of the top ipa canonical pathways significantly affected by exposure to ir. the identification of gene families, from the gsea molecular signatures database, gives a functional overview of the genes identified as significantly altered after ir. gene families share common features such as biochemical activity and homology. gene family analysis shows transcription factors as well as protein kinases whose expression levels are significantly altered after ir. this reflects the broad nature of the transcription program initiated by genotoxic dna damage. physiologic and genotoxic damage induce a shared lymphocyte-specific response in previous work, we identified a lymphocyte-specific pattern of gene expression in response to physiologic dsdna breaks induced by rag endonuclease cleavage in primary pre-b cells and abl pre-b cell lines. here we examined the similarities in gene expression changes in response to both physiologic and genotoxic damage. we identified probes representing genes that are significantly regulated in the same direction in response to both damage types. since our wt cells exposed to ir are wt for rag endonuclease, there may be a contribution of naturally occurring rag-induced breaks in addition to the ir-induced dna damage in these cells and the gene expression response may contain elements of responses to both types of dna lesions. however, we expect the contribution of the rag-induced breaks to be minor since both the control and irradiated cells should respond in a similar manner to the sti- and since these cells are also wt for the repair pathway machinery that rapidly resolves these breaks. the similarly regulated genes include many lymphocyte specific maturation genes such as cd cd swap and nfκb. while these genes are regulated in response to both damage types, one difference we observed was that genotoxic damage appears to induce a more robust change in many of the affected genes and pathways. ipa reveals affected canonical pathways consistent with b cell maturation. since cd expression plays an important role in b cell maturation and its mrna levels are increased after dna damage, we compared the protein expression of cd after physiologic and genotoxic damage by flow cytometric analysis. we observed the expected increase in the number of cells with increased cd surface expression 90 minutes after 1 gy ir and this is in agreement with our previously published data showing an increase in cell numbers with higher cd expression levels after rag-induced dna breaks. genotoxic damage, but not physiologic damage, induces a potential cancer susceptibility cellular response in addition to the similarities in response to both types of dna damage, we observed a robust gene expression profile after genotoxic damage that was not seen after the physiologic damage. we identified probes, representing almost unique genes, which were differentially regulated in the response to ir but not in the response to physiologically induced damage. the transcriptional response unique to the ir-induced damage includes increased expression of oncogenes, protein kinases and transcription factors, as well as decreased expression of tumor suppressors and transcription factors. changes in the expression in these broad-range signalling molecules suggest a diverse biological response to genotoxic dsbs. in order to understand the broad biological mechanism and pathways affected by ir-induced dsbs, we utilized ipa to investigate the changes in pathways and biological functions caused by genotoxic damage. several canonical pathways were affected in response to ir that were not seen to be affected in physiologic-induced profiles, such as an nrf2-mediated oxidative stress response and cell cycle regulation pathways. as mentioned above, we also see a stronger enrichment of pathways associated with activation of mature b cells in response to antigen. initial inspection of the genes significantly regulated by ir-induced damage revealed several oncogenes and tumor suppressor genes whose expression change correlates with changes reported to be involved in cancer formation. these included increased expression of known proto-oncogenes, such as kras and rras, and the oncomir microrna-, as well as suppression of the expression of socs a known tumor suppressor. while the affymetrix mouse genome genechip array is not specifically designed to recognize micrornas, the current annotation of the array revealed that several micrornas are represented in the array. microrna- is known to target socs and this combination of increased expression of mir- and suppression of socs has been described in several b cell-derived lymphomas. our analysis shows many biological functions affected that are consistent with over-expression of potential oncogenes, such as cancer formation, cellular proliferation, and cell-mediated immune responses. increased mir- expression suggests an increased capacity for cellular proliferation after ionizing radiation increased expression of mmu-mir- in response to ir-induced damage suggests an increase in the capacity for proliferation of developing b cells when exposed to genotoxic stress. mature and functional micrornas are processed through a series of steps from a primary microrna transcript. it is possible the change in the mmu-mir- levels seen on the microarray is a reflection in the change of the primary mir- transcript. in order to investigate the changes of the mature microrna levels after ir-induced dsbs and to further examine investigate the expression of mir- and its target socs we undertook a time course to analyze and validate the mir- response to genotoxic damage over time. we used quantitative real-time pcr to track the mir- and socs expression levels at and 8 hours post ionizing radiation in the wt lines. a robust increase of mir- expression levels is seen in the initial hours after genotoxic damage, with an average of and fold increases seen at and 4 hours, respectively, post ir. by 8 hours post ir it appears the levels of mir- are returning to baseline. in an inverse correlation, socs expression is robustly decreased initially after ir exposure and has almost returned to baseline levels by 8 hours post damage and additional file ). activation of nrf after ir-induced dna damage suggests a cellular protective response to oxidative stress one pathway we observed to be regulated after genotoxic but not physiologic damage was the nrf oxidative stress pathway. members of the nrf signalling pathway, including maff, sqstm and txnrd were up regulated after exposure to genotoxic dna damage by microarray. since ir exposure is known to induce oxidative stress, induction of this pathway was expected, but in combination with the potential for increased proliferation suggested by mir- expression, it may reflect a potentially dangerous, cancer predisposed situation in response to genotoxic damage. in order to investigate and validate the induction of an nrf response to ir, we utilized a time course post ir to examine investigate nrf protein expression levels as well as mrna expression levels of the downstream target txnrd in each of the wt pre-b lines. changes in nrf protein expression were determined by quantitation from western blots of the wt lines, with each nrf band adjusted for the loading control β-actin. once activated, nrf protein accumulates in the nucleus, where it transactivates numerous target genes. in response to ir exposure, nrf protein levels were found to be on average fold higher than in mock-irradiated controls at 2 hours post treatment. levels continue to increase, reaching a peak at 4 hours post ir exposure. levels of nrf are almost completely returned to baseline levels after 8 hours. one downstream target of nrf is thioredoxin reductase, which plays a role in protecting cells from oxidative stress. as expected, mrna levels of txnrd as measured by qrt-pcr and normalizing to 18s levels, are increased at both and 4 hours post genotoxic damage. by 8 hours the levels of txnrd are returning to normal levels. discussion in this study we evaluated the transcriptional response induced by physiologic and genotoxic dsbs in developing b cells. by comparing these different types of damage we found that there are important similarities as well as striking differences in the cellular responses to these different forms of dna lesions. in previous work, we observed a lymphocyte-specific response to physiologically generated rag-induced dsdna breaks. highlights of this response include changes in the expression of genes important in immune function and maturation. changes in these genes suggest an increase in the signalling of the cd and nfκb pathways, suggesting a role for dna breaks in the progression of b cell maturation. after the observation that rag-induced dsbs trigger a response to move the b cells toward maturation, the next obvious question is whether or not other types of dsbs induce the same lymphocyte-specific maturation profile. here we observed probes that were differentially regulated in the same direction after induction of breaks regardless of the source of the damage. these changes include increased expression of cd cd icam swap nfκb, as well as other immune related genes. increased expression of these genes and others associated with cd40-, cd69-, and nfκb-related pathways suggest that the b cells are preparing to undergo maturation irrespective of the source of the dna damage. while a core set of genes is regulated in the same direction after both types of damage, the response to genotoxic damage is generally more robust than the response to rag-induced breaks. we hypothesize this is due to the greater amount of dna damage induced by ir exposures, perhaps initiating a stronger signal towards maturation. while we see similarities in the response to physiologic and genotoxic breaks, we recognize there are potential biological and technical differences in comparing rag-induced and ionization radiation-induced dsbs. rag-induced dsbs are tightly regulated and only a small number of breaks in very specific locations in the genome are induced. in contrast, ir exposure can induce a broad range of dna damage, including dsbs, that is not restricted to specific locations but occurs throughout the genome and can involve dna lesions with damaged nucleotide ends as opposed to the “clean” ends generated by the rag endonucleases. we have attempted to mitigate these differences by using exposures to a low dosage of ir and by ensuring the cells are in the same phase of the cell cycle to ensure that similar dna damage repair processes would be available under both conditions. despite the differences in the nature of the dsbs it appears that the cells undergo a similar response to that damage by initiating a central lymphocyte-specific transcriptional response that is common to both. in addition to these similarities, the genotoxic damage also induces changes in unique probes representing almost genes. broad expression changes in transcription factors and protein kinases suggest genotoxic dsbs induce a myriad of changes in both gene expression and physiological pathways. gene expression changes and alterations in pathways associated with b cell activation, increased proliferation, and oxidative stress responses are seen in the unique response to ir. many of the pathways altered on a transcriptional level are known to be involved in the generation of cancers. also, this study highlights the importance of additional layers of regulation, which has become obvious with the discovery of genotoxic regulation of small regulatory rnas such as micrornas. their role in a wide variety of physiological processes has revealed their vital importance in proper cellular function, and disregulation has been linked to human diseases, including cancer and immune disorders. in addition to the lymphocyte specific transcriptional pattern induced by both types of dna damage, genotoxic damage induces a potentially oncogenic combination of alterations of genes and biological response pathways. we found that genotoxic, but not physiologic, damage induces increased expression of several proto-oncogenes such as kras and the oncomir mir- this suggested to us that the cellular response to double strand dna damage could be specific to the method of generation of that damage, recognizing that ir-induced genotoxic damage causes many types of dna damage. mir- is a known oncogenic mirna and its increase has been correlated with formation of b cell malignancies. the up-regulation of mir- at both the primary transcript and mature microrna level, seen after ir-induced breaks suggests a potential for the development of cancer after genotoxic damage. additionally, mir- has been identified to suppress a number of tumor suppressors, including socs which we found to be suppressed after genotoxic damage. mir- up-regulation has been associated with b cell cancers and b cell transformation, as well as with normal immune response. altered expression of this mirna and its target socs suggests that an increase in proliferation may be triggered after ir exposure. interestingly, this increase in proliferation and up-regulation of mir- has been seen in mature b cells as a result of their response to antigen. another noteworthy difference between genotoxic and physiologic damage is the significant change in regulation of genes in b cell activation pathways and nrf2-mediated signalling. nrf signalling is a known response to ir but has also been seen in the activation of mature b cells. the nrf pathway is a critical regulator of the defense against oxidative stress. activation of nrf pathways is an important component in the clearing of oxidative stress and in a cytoprotective outcome. there has been some suggestion that nrf also has a role in rescuing cells from cell cycle arrest that can be generated in response to oxidative damage. these responses to genotoxic damage in both the b cell activation and nrf signalling pathways could combine to result in serious deleterious consequences to the immune system.  the broad gene expression alterations, increased expression of the oncomir mir- and proto-oncogenes such as kras, the activation of nrf and the lymphocyte specific maturation profile induced by genotoxic dsbs, reflect a potentially dangerous combination of conflicting signals for increased cellular proliferation and cytoprotective responses. these conflicting signals could drive developing b cells to continue to mature and proliferate in the presence of dna damage after genotoxic exposures. the possibility of maturation and proliferation in the presence of dna damage increases the risk for aberrant repair or lack of repair of damaged dna. continued maturation and proliferation of these highly proliferative cells signalled by genotoxic dsbs provides a mechanism for the development of immunodeficiencies due to the potential loss of mature functional b cells as well as for the formation of lympho-proliferative cancers when dna repair is not completed successfully. 
double strand dna breaks are a form of dna damage that can be generated from both genotoxic exposures and physiologic processes, can disrupt cellular functions and can be lethal if not repaired properly. here we compare transcriptional responses to physiological dna breaks with responses to genotoxic dna damage induced by ionizing radiation. comparing these transcriptional responses provides a greater understanding of the mechanisms cells use in the differentiation between types of dna damage and the potential consequences of different sources of damage. mir-155b cellsionizing radiationdna damagedouble strand breakstranscriptome profiles physiologic dsdna breaks are generated in a variety of normal cellular functions, including the rag endonuclease-mediated rearrangement of antigen receptor genes during the normal development of lymphocytes. genotoxic damage causes robust alterations to pathways associated with b cell activation and increased proliferation, suggesting that genotoxic damage initiates not only the normal b cell maturation processes but also mimics activated b cell response to antigenic agents. notably, changes including elevated levels of expression of kras and mmu-mir- and the repression of socs were observed following genotoxic damage, reflecting induction of a cancer-prone phenotype. these results suggest genotoxic damage may induce a unique cancer-prone phenotype and processes mimicking activated b cell response to antigenic agents, as well as the normal b cell maturation processes. we identified a central lymphocyte-specific transcriptional response common to both physiologic and genotoxic breaks, which includes many lymphocyte developmental processes.
 double strand dna breaks are a form of dna damage that can be generated from both genotoxic exposures and physiologic processes, can disrupt cellular functions and can be lethal if not repaired properly. physiologic dsdna breaks are generated in a variety of normal cellular functions, including the rag endonuclease-mediated rearrangement of antigen receptor genes during the normal development of lymphocytes. we previously showed that physiologic breaks initiate lymphocyte development-specific transcriptional programs. here we compare transcriptional responses to physiological dna breaks with responses to genotoxic dna damage induced by ionizing radiation.  we identified a central lymphocyte-specific transcriptional response common to both physiologic and genotoxic breaks, which includes many lymphocyte developmental processes. genotoxic damage causes robust alterations to pathways associated with b cell activation and increased proliferation, suggesting that genotoxic damage initiates not only the normal b cell maturation processes but also mimics activated b cell response to antigenic agents. notably, changes including elevated levels of expression of kras and mmu-mir- and the repression of socs were observed following genotoxic damage, reflecting induction of a cancer-prone phenotype.  comparing these transcriptional responses provides a greater understanding of the mechanisms cells use in the differentiation between types of dna damage and the potential consequences of different sources of damage. these results suggest genotoxic damage may induce a unique cancer-prone phenotype and processes mimicking activated b cell response to antigenic agents, as well as the normal b cell maturation processes. mir-155b cellsionizing radiationdna damagedouble strand breakstranscriptome profiles 