S692 I. J. Radiation Oncology d Biology d Physics Volume 72, Number 1, Supplement, 2008

changed significantly: a rapid transition from low cytoplasmic perinuclear levels in unirradiated BxPC-3 and Panc-1 cells tohigh membrane levels shortly after radiation. Consistent results were obtained in nude mice bearing BxPC-3 tumors. Frozensections of unirradiated tumors, immunostained with fluorescent anti alpha 5 beta 1 and anti MMP 1 Abs, showed only lowlevels of perinuclear alpha 5 beta 1 and MMP 1; while cells of irradiated tumors had high membrane levels of both proteins.Radiation-induced alpha 5 beta 1 upregulation may be due to increased alpha 5 transcription, since RT-PCR analysis ofirradiated BxPC-3 cells showed a 5-fold induction of alpha 5 mRNA. NF-kappa B may mediate radiation-induced alpha 5transcription: a single dose in BxPC-3 or Panc-1 cells caused a significant upregulation of IkB-beta throughout the cytoplasm,followed immediately by nuclear localization. The time and radiation dosage required for IkB-beta induction and nuclearlocalization corresponded closely to those observed for alpha 5 upregulation. Since IkB-beta functions as a chaperone toassist in NF-kB nuclear import, radiation-induced invasion may occur via NF-kB mediated transcription of the alpha 5gene, as reported previously in LPS stimulated cells. Both radiation induced alpha 5 beta 1 upregulation and invasionwere prevented by the PHSCN peptide, an invasion inhibitor targeting activated beta 1 integrins and currently in Phase IIclinical trial.

Author Disclosure: D.L. Livant, None; H. Yao, None; Z. Zeng, None; E. Staszewski, None; A. Spalding, None.

3163 The Radiobiology of Modulated Radiotherapy: Experimental Evidence and Models for Tumour Response

N. Suchowerska1, M. Ebert2, D. McKenzie3, M. Jackson1, C. Milross1

1Royal Prince Alfred Hospital, Camperdown, Australia, 2Sir Charles Gairdner Cancer Centre, Western Australia, Australia,3University of Sydney, Sydney, Australia

Purpose/Objective(s): The inherent spatial modulation of dose in IMRT makes the expression of bystander effects apparent. Tem-poral modulation of the dose deposited in multiple small field segments introduces variability in cell survival by changing the repairdynamics. In this study we provide experimental evidence that local dose alone is not an accurate predictor of response in mod-ulated fields. We develop a framework to include non local effects into a tissue response model, parameterized by comparison withexperiment. From the model the relative proportions of direct and bystander cell death can be evaluated.

Materials/Methods: The cell survival following exposure to spatially modulated beams was determined using in vitro experi-ments with malignant melanoma (MM576) and non-small-cell lung (NCl-H460) cell lines. The cell survival in modulated fields,as determined by a clonogenic assay, was compared to the cell survival in the uniformly irradiated fields. The effects of temporallymodulated beams on cell survival and incidence of double strand DNA breaks were determined using the clonogenic and thegH2AX assay respectively. The utility of both assays was improved by the development and use of new automated countingand analysis software (CHiTA). Three models were developed to describe cell response. All three included a direct radiation in-teraction as the primary cell killing mechanism, described by a linear-quadratic response. A secondary response to a bystandersignal was included, generated by the direct action of radiation via a lethal (Model 1), lethal or non-lethal (Model 2), or non-lethal(Model 3) interaction with another cell. The models were fitted to the experimental results for the two cell lines, following uniformand non uniform exposure. The model parameter values were then derived.

Results: Both cell lines exposed to spatially modulated fields exhibited bystander responses. The bystander signal was identified bythe models as an important factor in determining the ultimate cell survival fraction. The experimental data was best fitted usingModels 1 and 2, suggesting that the cell response to bystander signals is likely to be a lethal event. The bystander componentof cell response was much greater in MM576 than NCl-H460. For all models, bystander response accounted for between 20%and 50% of total cell death.

Conclusions: We have developed a framework for a radiobiological model, which includes bystander effects. The model enablesus to quantify the relative amounts of direct and bystander induced cell death. Key parameters in the bystander response are signalproduction, signal transport and response to the signal. A difference between in vivo and in vitro cell response could be explained bydifferences in the transport parameter.

This work was supported by the NSW Cancer Council.Author Disclosure: N. Suchowerska, NSW Cancer Council, B. Research Grant; M. Ebert, NSW Cancer Council, B. ResearchGrant; D. McKenzie, NSW Cancer Council, B. Research Grant; M. Jackson, NSW Cancer Council, B. Research Grant; C. Milross,NSW Cancer Council, B. Research Grant.

3164 A High Throughput Automated Microscopy RNAi Screen for Modifiers of the DNA Damage Response to

Ionizing Radiation

S. R. Floyd1,2, M. E. Pacold3,2, E. J. Blake2, D. M. Sabatini2,4, M. B. Yaffe2,1

1Beth Israel Deaconess Medical Center, Boston, MA, 2Massachusetts Institute of Technology, Cambridge, MA, 3HarvardRadiation Oncology Program, Boston, MA, 4Whitehead Institute, Cambridge, MA

Purpose/Objective(s): The response to DNA damage plays an important role in protecting the genome from aberrant function. Anelaborate machinery has evolved to protect the genome from multiple types of damage, incurred either from environmental toxins,or from normal cellular processes such as transcriptional events or VDJ recombination. Current cancer therapy makes use of se-lective DNA damage in cancer cells to cause cell death, and the mechanisms of cell cycle arrest and repair are critical to tolerance oftherapy by normal tissues. Ionizing radiation is of particular interest as a DNA damaging agent, due to its physical properties thatallow anatomic precision in dose delivery. In order to identify novel components of the cellular response to ionizing radiation, wedeveloped an automated microscopy assay to interrogate the DNA damage repair pathway for use in high throughput screening.This assay uses novel digital image analysis techniques to create a unique dataset of multiple quantitative measurements on indi-vidual cells.

Materials/Methods: We used the U2OS human osteosarcoma cell line, which has a robust and well-characterized response toionizing radiation, as a model system to search for molecules that affect the DNA damage response. We developed an immuno-fluorescence assay that simultaneously monitors phosphorylation of histone H2AX, phosphorylation of histone H3, DNA contentand nuclear morphology, as well as apoptotic cleavage of caspase 3. This assay is adapted for use in 384 well plates and robotic

Proceedings of the 50th Annual ASTRO Meeting S693

acquisition of digital microscopy images, followed by automated digital image analysis using the CellProfiler image analysis pro-gram, developed by our collaborators at the Whitehead and Broad Institutes.

Results: We have applied this assay to a validated library of shRNA molecules via a lentiviral expression system developed by TheRNAi Consortium at the Broad Institute of M.I.T. and Harvard. Using this strategy, we demonstrated anticipated effects of RNAiinhibition of many known components of the DNA damage pathway. Additionally, we identified novel modifiers of the response toionizing radiation.

Conclusions: We identified novel components of the DNA damage response to ionizing radiation in the U2OS human osteosar-coma cell line using RNAi and a robust high throughput assay that monitors multiple outputs of the DNA damage response. Thisassay holds the potential for adaptation to multiple cell lines or primary tumor cells to identify components of the DNA damageresponse in a wide variety of tumor model systems.

Author Disclosure: S.R. Floyd, None; M.E. Pacold, None; E.J. Blake, None; D.M. Sabatini, None; M.B. Yaffe, None.

3165 SOCS3 Regulates p21 Expression and Cell Cycle Arrest in Response to Radiation-induced DNA Damage

J. C. Sitko1, B. K. Yeh1, M. Kim2, H. Zhou1, G. Takaesu3, A. Yoshimura3, W. H. McBride1, A. Jewett1, C. A. M. Jamieson1,N. A. Cacalano1

1UCLA School of Medicine, Los Angeles, CA, 2Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea,3Kyushu University, Fukuoka, Japan

Purpose/Objective(s): Genotoxic agents such as ionizing radiation trigger cell cycle arrest at the G1/S and G2/M checkpoints,allowing cells to repair damaged DNA before entry into mitosis. DNA damage-induced G1 arrest involves p53-dependent expres-sion of p21 (Cip1/Waf-1), which inhibits cyclin-dependent kinases and blocks S phase entry. While much of the core DNA damageresponse has been well-studied, other signaling proteins that interact with and modulate this response remain uncharacterized. Inthis study, we identify Suppressor of Cytokine Signaling-3 (SOCS3) as an important regulator of radiation-induced G1 arrest.

Materials/Methods: Wild-type and SOCS3-deficient fibroblasts were treated with a 10 Gy dose of gamma radiation. At 0, 6, 12,and 24 hours after irradiation, the cells were analyzed by propidium iodide flow cytometry, RT-PCR, immunoprecipitation, andWestern blot protein analysis.

Results: The SOCS3-deficient fibroblasts fail to undergo G1 arrest and accumulate in the G2/M phase of the cell cycle. SOCS3knockout cells phosphorylate p53 and H2AX normally in response to radiation, but fail to upregulate p21 expression. In addition,STAT3 phosphorylation is elevated in SOCS3-deficient cells compared to wild-type cells. Normal G1 arrest can be restored inSOCS3-deficient cells by retroviral transduction of wild-type SOCS3 or a dominant negative mutant of STAT3.

Conclusions: Our results suggest a novel function for SOCS3 in the control of genome stability by negatively regulating STAT3-dependent radioresistant DNA synthesis, and promoting p53-dependent p21 expression.

Author Disclosure: J.C. Sitko, None; B.K. Yeh, None; M. Kim, None; H. Zhou, None; G. Takaesu, None; A. Yoshimura, None;W.H. McBride, None; A. Jewett, None; C.A.M. Jamieson, None; N.A. Cacalano, None.

3166 MYC Potentiates DNA Damage Response Dependent Cellular Senescence

P. T. Tran, J. Chen, P. Choi, G. Horng, D. W. Felsher

Stanford Comprehensive Cancer Center, Stanford, CA

Purpose/Objective(s): Oncogenesis is the progression of a somatic cell through a finite number of phenotypic stages to be-coming a cancer. These stages are defined by genetic and epigenetic lesions that activate oncogenes and disable tumor sup-pressor functions. Cancer cells can exhibit genomic instability, but the true contribution of genomic instability to cancerdevelopment is unknown. Multiple DNA repair pathways ensure genomic integrity and recent evidence suggests that DNAdamage response (DDR) pathways serve as an initial barrier to oncogenic stress by inducing cell death or cellular senescence.Cellular senescence is a tumor suppressor mechanism in which precancerous cells enter a permanent state of quiescence.MYC is the prototypical oncogene that functions as a transcription factor to promote unrestrained cellular proliferation andblocks differentiation. Increasing evidence suggests that MYC may also contribute to oncogenesis by inducing genomicinstability.

Materials/Methods: Normal primary cells expressing an inducible form of MYC were treated with the DNA replication stressagent hydroxyurea (HU) and/or DDR inhibitors caffeine and UCN-01. Components of the DDR were assayed for phosphorylationstatus, change in mobility during electrophoresis and protein levels by Western analysis and immunofluorescence. Cell death andcellular senescence were performed using flow cytometry (FACs) and senescence-associated beta-galactosidase (SAB) staining,respectively.

Results: MYC augmented DDR signaling following treatment with HU as demonstrated by phospho-western of DDR compo-nents. MYC also increased cell death following HU treatment shown by sub-G1 DNA content and annexinV/7-AAD FACs. Sim-ilar induction of the DDR was not apparent for other forms of MYC-potentiated cell death not involving DNA damage. MYCpotentiating the DDR after treatment with HU was intriguing in light of data suggesting that Ras and BRAF oncogenes cause in-creased DNA replicational stress resulting in oncogene-induced senescence (OIS) dependent on the DDR. MYC increased numbersof senescent cells using the marker SAB in the presence of HU. Inhibitors of the DDR prevented MYC-dependent increase incellular senescence, but caused a concomitant increase in cell death.

Conclusions: We have demonstrated for the first time that MYC, like the oncogenes Ras and BRAF, induce senescence in normalprimary cells. Recent evidence has linked MYC to DNA replication stimulation at the post-translational level. Our data is sugges-tive that MYC promotes DNA replicational stress thus exciting the DDR signaling pathway and leading to OIS and/or cell death.Taken together our data suggests that OIS may be a general phenomenon resulting from increased DNA replication stress.

Author Disclosure: P.T. Tran, None; J. Chen, None; P. Choi, None; G. Horng, None; D.W. Felsher, None.