Project description:Proton irradiation is touted for its improved tumor targeting due to the physical advantages of ion beams for radiotherapy. Recent studies from our laboratory have shown that, in addition to targeting advantages, proton irradiation can inhibit angiogenic and immune factors and thereby modulate tumor progression. High-energy protons also constitute a principal component of the galactic cosmic rays to which astronauts are exposed. Increased understanding of the biological effects of proton exposure would thus contribute to both improved cancer therapy and carcinogenesis risk assessment for space travel. In addition, age plays a major role in tumor incidence and is a critical consideration for estimating cancer risk. We investigated the effects of host age and proton exposure on tumor progression. Tumor lag time and growth dynamics were tracked following injection of murine Lewis lung carcinoma (LLC) cells into young (68 day) versus old (736 day) mice with or without coincident irradiation. Tumor progression was suppressed in old compared to young mice. Differences in progression were further modulated by proton irradiation (1GeV), with increased inhibition evident in old mice. Through global transcriptome analysis, TGFÎ²1 and TGFÎ²2 were determined to be key players that contributed to the tumor dynamics observed. These findings point to older hosts providing decreased systemic tumor support, which can be further inhibited by proton irradiation. For genome-wide expression profiling of tumor tissue, Mouse WG-6 BeadArray chips (Illumina, San Diego, CA) were used. Total RNA was amplified with the Ambion Illumina TotalPrep Amplification Kit (Ambion, Austin, TX) and labeled from all replicate biological samples for each condition. For tumor replicates, thirty tumor samples from adolescent and thirty tumor samples from old mice, for a total of 60 tumor samples, were used. All replicate samples were run individually. For each age group, ten tumor samples had received proton irradiation while twenty tumor samples were from unirradiated mice (as described above). Total RNA was isolated and purified using TRIzol (Invitrogen) and quantified using an Agilent Bioanalyzer. Samples were deemed suitable for amplification and hybridization if they had 28s/18s = 2:1, RIN >7. Total RNA of 500ng per sample was amplified using AmbionTotalPrep, and 1.5ug of the product was loaded onto the chips. Following hybridization at 55C, the chips were washed and then scanned using the Illumina iScan System. The data was checked with GenomeStudio (Illumina) for quality control. In GenomeStudio, data was background subtracted and rank invariant normalization was applied. Data was imported into MultiExperiment Viewer, MeV, for statistical analysis. The statistically significant genes were determined using MeV by applying a one-way ANOVA analysis with standard Bonferroni correction with a FDR <0.05 that resulted in a list of significant genes. Average gene expression signals <10 were filtered out due to signal being

Project description:Age plays a crucial role in the interplay between tumor and host; with further perturbations induced by irradiation. Proton irradiation on tumors induces biological modulations including inhibition of angiogenic and immune factors critical to “hallmark” processes impacting tumor development, in addition to physical targeting advantages. These advantages have provided promising results for proton therapy in cancer. Additionally, protons have implications for carcinogenesis risk of space travel (due to the high proportion of high energy protons in space radiation). Through a systems biology approach, we investigated how host tissue (i.e. splenic tissue) of tumor-bearing mice is altered with age, with or without whole-body proton exposure. Transcriptome analysis was performed on splenic tissue from adolescent (68 day) versus old (736 day) C57BL/6 male mice injected with Lewis lung carcinoma cells with or without three fractionations of 0.5Gy (1GeV) proton irradiation. Global transcriptome analysis indicated that proton irradiation of adolescent hosts caused significant signaling changes within splenic tissues that support carcinogenesis within the mice, as compared to old subjects. Increases in cell cycling and immunosuppression in irradiated adolescent hosts with CDK2, MCM7, CD74, and RUVBL2 as the key players were involved in the regulatory changes in host environment response (i.e. spleen). These results suggest a significant biological component to proton irradiation, operative through host age, that would indicate a modulation of host’s ability to support carcinogenesis in adolescence and the bestowal of resistance to immunosuppression, carcinogenesis, and genetic perturbation by old age. For genome-wide expression profiling of tumor tissue, Mouse WG-6 BeadArray chips (Illumina, San Diego, CA) were used. Total RNA was amplified with the Ambion Illumina TotalPrep Amplification Kit (Ambion, Austin, TX) and labeled from all replicate biological samples for each condition. For spleen replicates, 9 spleen samples from adolescent with 0Gy , 10 from adolescent with 0.5Gyx3 protons, 9 from old from old with 0Gy, and 10 from old mice with 0.5Gyx3 protons, were used. All replicate samples were run individually. Total RNA was isolated and purified using TRIzol (Invitrogen) and quantified using an Agilent Bioanalyzer. Samples were deemed suitable for amplification and hybridization if they had 28s/18s = 2:1, RIN >7. Total RNA of 500ng per sample was amplified using AmbionTotalPrep, and 1.5ug of the product was loaded onto the chips. Following hybridization at 55C, the chips were washed and then scanned using the Illumina iScan System. The data was checked with GenomeStudio (Illumina) for quality control. Data were corrected through COMBAT correction, quantile normalized, collapsed to genes from probes, then imported into MultiExperiment Viewer, MeV for analysis. Statistically significant genes were determined by applying a one-way ANOVA with an adjusted Bonferroni correction and false discovery rate (FDR) < 0.001 that resulted in a list of significant genes.

Project description:Age plays a major role in tumor incidence and is an important consideration when modeling the carcinogenesis process or estimating cancer risks. Epidemiological data show that from adolescence through middle age, cancer incidence increases with age. This effect is commonly attributed to a lifetime accumulation of cellular, particularly DNA, damage. However, during middle-age, the incidence begins to decelerate and, for many tumor sites, it actually decreases at sufficiently advanced ages. We investigated if the observed deceleration and potential decrease in incidence could be attributed to a decreased capacity of older hosts to support tumor progression, and whether HZE (high atomic number (Z), high energy (E)) radiation differentially modulates tumor progression in young versus middle-age hosts, issues relevant to estimating carcinogenesis risk for astronauts. Lewis lung carcinoma (LLC) cells were injected into syngeneic mice (143 and 551 days old), which were then subject to whole-body 56Fe irradiation (1GeV/amu). Three findings emerged: 1) among unirradiated animals, substantial inhibition of tumor progression and significantly decreased tumor growth rates were seen for middle-aged mice compared to young mice; 2) whole-body 56Fe irradiation (1GeV/amu) inhibited tumor progression in both young and in middle-aged mice (with greater suppression seen in case of young animals), with little effect on tumor growth rates; and 3) 56Fe irradiation (1GeV/amu) suppressed tumor progression in young mice, to a degree not significantly different than transiting from young to middle-aged. Thus, 56Fe irradiation (1GeV/amu) acted similar to aging with respect to tumor progression. We further investigated the molecular underpinnings driving the radiation modulation of tumor dynamics in young and middle-aged mice. Through global gene expression analysis, the key players, FASN, AKT1, and the CXCL12/CXCR4 complex, were determined to be contributory. In sum, these findings demonstrate a reduced capacity of middle-aged hosts to support the progression phase of carcinogenesis and identify molecular factors contributory to HZE radiation modulation of tumor progression as a function of age.

Project description:Age plays a major role in tumor incidence and is an important consideration when modeling the carcinogenesis process or estimating cancer risks. Epidemiological data show that from adolescence through middle age, cancer incidence increases with age. This effect is commonly attributed to a lifetime accumulation of cellular, particularly DNA, damage. However, during middle-age, the incidence begins to decelerate and, for many tumor sites, it actually decreases at sufficiently advanced ages. We investigated if the observed deceleration and potential decrease in incidence could be attributed to a decreased capacity of older hosts to support tumor progression, and whether HZE (high atomic number (Z), high energy (E)) radiation differentially modulates tumor progression in young versus middle-age hosts, issues relevant to estimating carcinogenesis risk for astronauts. Lewis lung carcinoma (LLC) cells were injected into syngeneic mice (143 and 551 days old), which were then subject to whole-body 56Fe irradiation (1GeV/amu). Three findings emerged: 1) among unirradiated animals, substantial inhibition of tumor progression and significantly decreased tumor growth rates were seen for middle-aged mice compared to young mice; 2) whole-body 56Fe irradiation (1GeV/amu) inhibited tumor progression in both young and in middle-aged mice (with greater suppression seen in case of young animals), with little effect on tumor growth rates; and 3) 56Fe irradiation (1GeV/amu) suppressed tumor progression in young mice, to a degree not significantly different than transiting from young to middle-aged. Thus, 56Fe irradiation (1GeV/amu) acted similar to aging with respect to tumor progression. We further investigated the molecular underpinnings driving the radiation modulation of tumor dynamics in young and middle-aged mice. Through global gene expression analysis, the key players, FASN, AKT1, and the CXCL12/CXCR4 complex, were determined to be contributory. In sum, these findings demonstrate a reduced capacity of middle-aged hosts to support the progression phase of carcinogenesis and identify molecular factors contributory to HZE radiation modulation of tumor progression as a function of age. For genome-wide expression profiling of tumor tissue, Mouse WG-6 BeadArray chips (Illumina, San Diego, CA) were used. Total RNA was amplified with the Ambion Illumina TotalPrep Amplification Kit (Ambion, Austin, TX) and labeled from all replicate biological samples for each condition. The number of tumor sample replicates used from each condition is as follows: 10 samples from young unirradiated mice, 8 samples from young irradiated mice, 7 samples from middle-aged unirradiated mice, 5 samples from middle-aged irradiated mice. Total RNA was isolated and purified using Trizol (Invitrogen) or RNeasy (Qiagen), quantified and qualified using Agilent Bioanalyzer (Agilent) and samples were deemed suitable for amplification and hybridization if they had O.D. 260/280 = 1.7 - 2.1, 28s/18s = 2:1, RIN (RNA integrity number) >7. Total RNA of 500ng per sample was amplified using Ambion TotalPrep (Ambion), and 1.5Âµg of the product was loaded onto the chips. Following hybridization at 55C, the chips were washed and then scanned using the Illumina iScan (Illumina), and the data were analyzed using GenomeStudio (Illumina). Data were first analyzed for gene expression and then culled for present genes (genes that meet the criteria of detection p-value < 0.05). Expression above background was included in an expressed genes working data set for further analyses. Rank variant normalization was applied to the data before extensive analysis. Differential gene expression analysis was used to compare to the reference group, young unirradiated mice, and genes were then evaluated and validated.

Project description:Children are considered more sensitive to radiation-induced cancer than adults, yet any differences in genomic alterations associated with age at exposure and their underlying mechanisms remain unclear. We analyzed the mutation of key tumor suppressor genes in T-cell lymphomas arising after weekly irradiation of female B6C3F1 mice with 1.2 Gy X-rays for 4 consecutive weeks starting during infancy (1 week old), adolescence (4 weeks old) or as young adults (8 weeks old). T-cell lymphoma incidence did not show any statistical differences among three irradiated groups; however, the frequency of loss of heterozygosity on chromosomes 11 and 19 showed opposing correlations with age at exposure. Ikaros on chromosome 11 was mutated frequently in the young adultâ??irradiation group (63%; 5/8), frequently incurring multiple independent hits (including deletions and mutations) or suffering a single hit predicted to result in a dominant negative protein (such as those lacking exon 4, an isoform we have designated Ik11, which lacks two of the four zincâ??finger domains important for DNA binding). Pten on chromosome 19 was mutated more frequently in tumors from infant- (67%; 10/15) compared with young adult-irradiated mice (13%; 1/8, P = 0.041). Despite the presence of homozygous Pten mutations, DNA copy number was unchanged in the infant-irradiation group, suggesting duplication of the mutated allele by chromosome mis-segregation or mitotic recombination. Taken together, our findings demonstrate that while deletions on chromosome 11 affecting the Ikaros locus are a prominent feature of young adult irradiation-induced T-cell lymphoma, tumors arising after infant irradiation suffer a second hit in the Pten gene by chromosome mis-segregation or recombination. This is the first report showing age-at-exposure associated radiation-induced genomic alterations in two key tumor suppressor genes linked to T-cell lymphomagenesis. These data are important for considering the risks associated with childhood exposure to radiation. 38 T-cell lymphomas arising after weekly irradiation of female B6C3F1 mice with 1.2 Gy X-rays for 4 consecutive weeks starting at different ages (Infant: 1 week old (15 tumors), Adolescent: 4 weeks old (13 tumors), Young adult: 8 weeks old (10 tumors)) were analyzed.

Project description:Children are considered more sensitive to radiation-induced cancer than adults, yet any differences in genomic alterations associated with age at exposure and their underlying mechanisms remain unclear. We analyzed the mutation of key tumor suppressor genes in T-cell lymphomas arising after weekly irradiation of female B6C3F1 mice with 1.2 Gy X-rays for 4 consecutive weeks starting during infancy (1 week old), adolescence (4 weeks old) or as young adults (8 weeks old). T-cell lymphoma incidence did not show any statistical differences among three irradiated groups; however, the frequency of loss of heterozygosity on chromosomes 11 and 19 showed opposing correlations with age at exposure. Ikaros on chromosome 11 was mutated frequently in the young adult–irradiation group (63%; 5/8), frequently incurring multiple independent hits (including deletions and mutations) or suffering a single hit predicted to result in a dominant negative protein (such as those lacking exon 4, an isoform we have designated Ik11, which lacks two of the four zinc–finger domains important for DNA binding). Pten on chromosome 19 was mutated more frequently in tumors from infant- (67%; 10/15) compared with young adult-irradiated mice (13%; 1/8, P = 0.041). Despite the presence of homozygous Pten mutations, DNA copy number was unchanged in the infant-irradiation group, suggesting duplication of the mutated allele by chromosome mis-segregation or mitotic recombination. Taken together, our findings demonstrate that while deletions on chromosome 11 affecting the Ikaros locus are a prominent feature of young adult irradiation-induced T-cell lymphoma, tumors arising after infant irradiation suffer a second hit in the Pten gene by chromosome mis-segregation or recombination. This is the first report showing age-at-exposure associated radiation-induced genomic alterations in two key tumor suppressor genes linked to T-cell lymphomagenesis. These data are important for considering the risks associated with childhood exposure to radiation.

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Proton irradiation augments the reduction in tumor progression observed with advanced age

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