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specific CD8+ T cells. Results: TSA tumors were resistant to PD-1 blockade. RT delayed tumor growth
(p<0.01), but regression was seen only in 1 of 6 mice. In contrast, all mice given RT+RMP1-14 completely
rejected tumors by day 25. Splenic AH1-specific CD8+ T-cells were markedly increased (4.6%) with
RT+RMP1-14 compared to control (1.7%), RMP1-14 (1.8%) or RT (2.9%) treated mice (p<0.05). CD8+ T
cells expressing activation markers CD69 and CD137 were increased in tumors of mice treated with RT
(64%) compared to control (42%) (p<0.001) irrespective of the treatment with RMP1-14. However, RT-
treated mice showed increase in CD8+ T cells expressing high levels of PD-1 (67% in RT vs 36% in control,
p<0.01). RT upregulated PDL-1 and PDL-2 on TSA cells and myeloid cells that limit activity of PD-1+ CD8+
T cells. Conclusion: Results demonstrate that countering negative signals by PD-1 improve RT-induced
tumor-specific CD8 T cells cross-priming and tumor rejection. The relative contribution of improved cross-
priming and effector function in the tumor by blocking PD-1 remain to be defined. However, these data
support testing this combination in the clinic.
(PS3-38) Altered microRNA expression pattern after single and fractionated radiation in prostate
carcinoma cells. Patricia Rivera; Molykutty J-Aryankalayil; Adeola Makinde; Sanjeewani Palayoor; and
Norman Coleman, NCI, Bethesda, MD
To understand the role of microRNAs (miRNA) in regulation of radiation-induced gene expression
and to help define potential radiation-inducible targets, miRNA expression was studied in LNCaP (p53
wild-type), PC3 (p53 null) and DU145 (p53 mutant). Methods: Cells were exposed to 10Gy as either a
single-dose (SD) radiation or multi-fractionated (MF) radiation. Microarray analyses were done using
human Agilent miRNA Microarray Kit (V2). Data were analyzed using Gene Spring software. Validation of
the miRNA expression and gene expression of miRNA targets was evaluated by real-time RT-PCR and
western blot analysis. Target filter analysis of differentially expressed miRNAs (>1.5 fold change and p
value<0.05) and their mRNA targets were analyzed. Results: Microarray analyses revealed that radiation
differentially expressed 84, 68 and 8 miRNAs with high confidence (>1.5fold change, p<0.05) in LNCaP,
PC3 and DU145 cells, respectively. MF radiation, compared to SD radiation exposure, induced more
miRNAs in all cell lines. In LNCaP and PC3 cells, fractionated radiation upregulated tumor suppressor
microRNAs miR-34a, miR-200, miR-135, miR-221 and let7. Baseline expression of miR-34a was markedly
reduced in PC3 cells and DU145 cells compared to LNCaP cells. However, miR-34a was upregulated by
fractionated irradiation in LNCaP and PC3 cells, but not in DU145 cells. RT-PCR analysis of 22
experimentally verified miR-34a targets showed distinct inverse correlation patterns in PC3 and LNCaP
cells at 6 and 24 hours after radiation. Inverse correlation of several miR-34a direct targets, such as BCL2,
CyclinD1, and Cdk4 were confirmed by Western Blot analysis. The majority of the differences in the
expression patterns were seen at later time points 24h after the final dose of radiation. Conclusion: Tumor
suppressor miR-34a was upregulated by fractionated radiation in radiosensitive LNCaP (p53 positive) and
PC3 (p53-null) cells indicating that radiation-induced miRNA expression may not be regulated by p53
alone. We are currently in the process of evaluating radiation-induced differential gene expression
changes by a combined approach of mRNA, miRNA and protein array analysis to identify radiation induced
molecular targets for cancer therapy. This work was supported by the Intramural Research Program of
the NIH, NCI, and CCR.
209 | P a g e
(p<0.01), but regression was seen only in 1 of 6 mice. In contrast, all mice given RT+RMP1-14 completely
rejected tumors by day 25. Splenic AH1-specific CD8+ T-cells were markedly increased (4.6%) with
RT+RMP1-14 compared to control (1.7%), RMP1-14 (1.8%) or RT (2.9%) treated mice (p<0.05). CD8+ T
cells expressing activation markers CD69 and CD137 were increased in tumors of mice treated with RT
(64%) compared to control (42%) (p<0.001) irrespective of the treatment with RMP1-14. However, RT-
treated mice showed increase in CD8+ T cells expressing high levels of PD-1 (67% in RT vs 36% in control,
p<0.01). RT upregulated PDL-1 and PDL-2 on TSA cells and myeloid cells that limit activity of PD-1+ CD8+
T cells. Conclusion: Results demonstrate that countering negative signals by PD-1 improve RT-induced
tumor-specific CD8 T cells cross-priming and tumor rejection. The relative contribution of improved cross-
priming and effector function in the tumor by blocking PD-1 remain to be defined. However, these data
support testing this combination in the clinic.
(PS3-38) Altered microRNA expression pattern after single and fractionated radiation in prostate
carcinoma cells. Patricia Rivera; Molykutty J-Aryankalayil; Adeola Makinde; Sanjeewani Palayoor; and
Norman Coleman, NCI, Bethesda, MD
To understand the role of microRNAs (miRNA) in regulation of radiation-induced gene expression
and to help define potential radiation-inducible targets, miRNA expression was studied in LNCaP (p53
wild-type), PC3 (p53 null) and DU145 (p53 mutant). Methods: Cells were exposed to 10Gy as either a
single-dose (SD) radiation or multi-fractionated (MF) radiation. Microarray analyses were done using
human Agilent miRNA Microarray Kit (V2). Data were analyzed using Gene Spring software. Validation of
the miRNA expression and gene expression of miRNA targets was evaluated by real-time RT-PCR and
western blot analysis. Target filter analysis of differentially expressed miRNAs (>1.5 fold change and p
value<0.05) and their mRNA targets were analyzed. Results: Microarray analyses revealed that radiation
differentially expressed 84, 68 and 8 miRNAs with high confidence (>1.5fold change, p<0.05) in LNCaP,
PC3 and DU145 cells, respectively. MF radiation, compared to SD radiation exposure, induced more
miRNAs in all cell lines. In LNCaP and PC3 cells, fractionated radiation upregulated tumor suppressor
microRNAs miR-34a, miR-200, miR-135, miR-221 and let7. Baseline expression of miR-34a was markedly
reduced in PC3 cells and DU145 cells compared to LNCaP cells. However, miR-34a was upregulated by
fractionated irradiation in LNCaP and PC3 cells, but not in DU145 cells. RT-PCR analysis of 22
experimentally verified miR-34a targets showed distinct inverse correlation patterns in PC3 and LNCaP
cells at 6 and 24 hours after radiation. Inverse correlation of several miR-34a direct targets, such as BCL2,
CyclinD1, and Cdk4 were confirmed by Western Blot analysis. The majority of the differences in the
expression patterns were seen at later time points 24h after the final dose of radiation. Conclusion: Tumor
suppressor miR-34a was upregulated by fractionated radiation in radiosensitive LNCaP (p53 positive) and
PC3 (p53-null) cells indicating that radiation-induced miRNA expression may not be regulated by p53
alone. We are currently in the process of evaluating radiation-induced differential gene expression
changes by a combined approach of mRNA, miRNA and protein array analysis to identify radiation induced
molecular targets for cancer therapy. This work was supported by the Intramural Research Program of
the NIH, NCI, and CCR.
209 | P a g e
