irradiation. Gene array technology is one method of measuring the expression of thousand of genes following irradiation,although the technology is not without problems in signal analyses and data interpretation. Given the complex interrelationshipamong gene expression patterns following irradiation, the relative degrees of gene expression in defined sub-groups, ascompared to the expression of a much larger number of genes, comprises a profile of differential gene expression. Because ofour interest in differential gene expression as mechanism modulating radiation induced cell survival, genetic profiles before andafter irradiation were compared in a radiobiologically well-characterized cell line, HELA.

Materials/Methods: HELA cells were cultured, using standard tissue culture techniques, under oxic conditions and exponentialgrowth phase. Cells were irradiated, using doses of 2 Gy, 5 Gy, and 10 Gy. Plates were trypsinized, cells washed twice in coldPBS, and the clean cell pellet was used for RNA extraction using Qiagen RNeasy extraction kit. Following RNA purification,the RNA was hybridized onto the HG-133 Affymetrix-based gene chip. A fluorescence scanner (Affymetrix GenChip System)was used to measure relative expression levels. Only genes with levels of expression that were differentially expressed two-foldin magnitude were considered genetic candidates for further studies.

Results: Interestingly, three genes were differentially expressed using the above criteria: dkk1 (an upstream regulator of wnt-1),itgb5 (an integrin closely related with itgb3, but rather poorly characterized in the literature), and rhogdi (a regulator ofRho-GTPase, a protein downstream of wnt-1, and regulated by integrin beta1). rhogdi and itgb5 were down-regulated followingirradiation and dkk1 was up-regulated. These changes were found as early as 2 hours of irradiation with the clinically relevant2 Gy fraction size.

Conclusions: Cell surface adhesion molecules, integrin beta5 and rho-GDI, are invoked in the formation of downstreameffectors of wnt-1 function. Interestingly, increased expression of dkk1, an upstream regulator of wnt-1, inhibits cyclin D1function and prolongs G1 arrest. In turn, an increase in radiation-induced apoptosis may result from the prolongation of the G1/Sinterval. In turn, decreased expression of both itgb5 and rhogdi, downstream of wnt-1, may result in decreases in cell surfaceadhesion protein, prolonged G1 arrest and corresponding increases in radiation-induced apoptosis. Further studies are neededusing additional cell lines in order to determine the generality of these findings with respect to the modulation of radiation-induced apoptosis following irradiation. On a more ambitious basis, the functional analyses of these proteins viz. radiationinduced cell survival may lead to the derivation of data predictive of radiotherapeutic response.

2057 Prediction of Radiotherapy-Induced Dermatitis by Comet Assay in Ex Vivo Irradiated Patients’Lymphocytes

N. Oya,1 H. Sai,1 S. Tachiiri,1 M. Mitsumori,1 Y. Nagata,1 M. Hiraoka1

1Department of Therapeutic Radiology and Oncology, Kyoto University Graduate School of Medicine, Kyoto, Japan

Purpose/Objective: It is estimated that approximately 5–10% of patients develop Grade 2 acute dermatitis following wholebreast radiotherapy as a part of breast conservative therapy for early breast cancer. The aim of the present study was to assessthe predictive value of ex vivo comet assay using patients’ lymphocytes for the occurrence of radiotherapy-induced dermatitis.

Materials/Methods: Among over 1,500 patients who had been treated with whole breast radiotherapy in our institute, 63patients entered this study. Twenty-nine patients who had developed Grade 2 whole breast bright erythema and 34 controlpatients were carefully selected, so that the patients characteristics and the therapeutic parameters of the two groups should beevenly matched. The patients’ lymphocytes were irradiated with a dose of 12 Gy, and fixed 0 to 180 minutes after irradiation.The cells were subjected to the single cell gel alkaline electrophoresis assay (comet assay), stained with propidium iodide, andprocessed by a fluorescence microscopic image analysis system to measure the amount of radiation-induced DNA damage.Standardized tail moment (STM) was determined at each time point, and a time-course curve was drawn for individual patients.

Results: In most cases, STM decreased with time, representing the repair procedure of radiation-induced DNA damage. Fourindependent DNA damage and repair parameters were calculated from the time-course curve; initial damage (ID) defined as theSTM at 0 min, residual damage (RD) defined as the STM at 180 min, repair half-time (RHT) defined as the time required toreduce the STM to 50% of the initial STM, and total repair capacity (TRC) defined as the area under the time-course curve ofSTM. The mean � SE of ID in the dermatitis group and the control group was 33.0 � 2.3 and 36.9 � 2.2 (p � 0.22), RD was7.3 � 1.1 and 7.2 � 0.9 (p � 0.94), RHT was 31.0 � 4.8 min and 20.6 � 2.5 min (p � 0.05), and TRC was 1860 � 190 and2080 � 160 (p � 0.37), respectively. The patients in the dermatitis group showed significantly longer RHT, while there wasno correlation between the other parameters and the occurrence of dermatitis.

Conclusions: The results suggested that RHT, a parameter representing DNA repair velocity in ex vivo comet assay, may bea candidate for the radiation dermatitis predictor in patients receiving whole breast radiotherapy. Further evaluation of this assayin a prospective setting is indicated.

2058 Does the Proinflammatory Cytokine, IL-1, Kick-Start the Pathway that Leads to Pulmonary LateRadiation Effects

J. P. Williams,1 E. Hernady,1 M. Levitt,1 C. Johnston,2 C. Reed,2 P. Okunieff,1 J. N. Finkelstein2,1

1Radiation Oncology, University of Rochester Medical Center, Rochester, NY, 2Pediatrics and Neonatology, University ofRochester Medical Center, Rochester, NY

Purpose/Objective: Classically, studies in different animal models have shown that inflammation and fibrosis representsignificant components of the spectrum of lung responses to insult. The specific hypothesis addressed in this study was thatradiation pulmonary injury induces early and persistent expression of interleukin (IL)-1. In particular, we believe that theexpression of members of this critical gene family in the initial injury/repair phase leads to changes in parenchymal cell cytokineand chemokine gene expression, resulting in the recruitment and activation of mononuclear cells. Once activated, the immuneand inflammatory cells release potent mitogenic and fibrogenic factors that, in combination with epithelial-derived factors,result in increased fibroblast proliferation and collagen biosynthesis. Using a specific gene ablation model, we examined theinteractions between IL-1 receptor expression and downstream chemokine expression and correlated these with cell recruitment,and the ultimate development of radiation pneumonitis and fibrosis.

S370 I. J. Radiation Oncology ● Biology ● Physics Volume 60, Number 1, Supplement, 2004

Materials/Methods: Using our standard experimental protocol for radiation-induced pulmonary injury, we irradiated groupsof IL-1R1-/- mice using 12.5 Gy whole thorax irradiation. Weights and breathing rates were measured weekly. The animalswere sacrificed at various time points post-lung irradiation. At these times, the lungs underwent bronchoalveolar lavage (BAL)and cell differentials were made. In addition, lungs were taken for mRNA and protein analysis by RNase protection assay andELISA, respectively. Lung sections were also taken for histologic and immunohistochemical processing followed by imageanalysis.

Results: At 10 weeks post-irradiation, a time point chosen since it represents the beginning of the infiltrative pneumonitic phasein wild-type C57Bl/6 mice, the IL-1R1-/- mice were sacrificed. Surprisingly, we found that the inflammatory cell numbers werehigher in the knockout animals than in the controls. This was confirmed by histologic examination and immunocytochemicalstaining, which demonstrated an increased state of inflammation in the knockout animals compared to control wild-types.However, by 30 weeks post-radiation, a period demonstrating high levels of fibrosis in wild-type animals, preliminary analysisappears to show little difference between knockouts and controls.

Conclusions: Results from early time points confounded our hypothesis for a leading role for the interleukin family in thedevelopment of radiation-induced pulmonary effects; these are now supported by data from later time points. However, theseresults may in fact indicate the measure of redundancy in the radiation injury response pathways. Such phenomena and possiblecandidates for alternative effectors will be discussed further.

This work was supported by NIH CA 11051–30.

2059 Applicability of Combination With the Vascular Targeting Agent ZD6126 in Boron Neutron CaptureTherapy

S. Masunaga,1 Y. Sakurai,1 K. Nagata,1 M. Suzuki,1 Y. Kinashi,1 K. Ono1

1Research Reactor Institute, Kyoto University, Osaka, Japan

Purpose/Objective: The aim of this study was to evaluate the antitumor efficacy of the vascular targeting agent ZD6126(N-acetylcochinol-O-phosphate) in the rodent SCC VII carcinoma model, in combination with boron neutron capture therapy(BNCT).

Materials/Methods: Sodium borocaptate-10B (BSH, 125 mg/kg, i.p.) or dl-p-boronophenylalanine-10B (BPA, 250 mg/kg, i.p.)as 10B-carrier was injected into SCC VII tumor-bearing mice, and 20 min later, ZD6126 (100 mg/kg, i.p.) was administered.Then the 10B concentrations in tumors and normal tissues were measured by prompt gamma-ray spectrometry. On the otherhand, for the thermal neutron beam exposure experiment, SCC VII tumor-bearing mice were continuously given 5-bromo-2’-deoxyuridine (BrdU) to label all proliferating (P) cells in the tumors, followed by treatment with a 10B-carrier and ZD6126 inthe same manner as the above-mentioned 10B pharmacokinetics analyses. To obtain almost similar intratumor 10B concen-trations during neutron exposure, thermal neutron beam irradiation was started from the time point of 30 min after injection ofBSH only, 90 min after BSH injection for combination with ZD6126, 120 min after the injection of BPA only, and 180 minafter BPA injection for combination with ZD6126. Right after the irradiation, the tumors were excised, minced and trypsinized.The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus(MN) frequency in cells without BrdU labeling (� quiescent (Q) cells) was determined using immunofluorescence staining forBrdU. Meanwhile, the MN frequency in total (P plus Q) tumor cells was determined from the tumors that were not pretreatedwith BrdU. The clonogenic cell survival assay was also performed in tumors from the mice given no BrdU.

Results: Pharmacokinetics analyses showed that combination with ZD6126 greatly increased the 10B concentrations in tumorsafter 60 min following BSH injection and after 120 min following BPA injection. The concentrations of 10B from BSH innormal tissues were also raised by combination with ZD6126, although not so clearly as those in tumors. Combination withZD6126 had almost no effect on the concentrations of 10B from BPA in normal tissues. The surviving fractions of total tumorcells and the MN frequencies of both total and Q tumor cells were reduced and increased by combination with ZD6126,respectively, whether BSH or BPA was employed. However, the degrees of these changes in the surviving fractions and the MNfrequencies were more obviously observed in tumors from BSH-injected mice than from BPA-injected mice, and in Q tumorcells than in total tumor cells whichever 10B-carrier was used.

Conclusions: Combination with ZD6126 was regarded as more promising in BSH-BNCT than in BPA-BNCT, and moreeffective for enhancing the sensitivity of intratumor Q cells than the total tumor cells. This induced the decrease in the extendeddifference in the sensitivity between the total and Q tumor cells caused by the use of 10B-carrier for BNCT.

2060 Incorporating Biological Metabolite Information Within Treatment of Prostate Carcinoma

E. K. Lee,1 M. Zaider2

1Center for Operations Research in Medicine, Georgia Tech, Atlanta, GA, 2Memorial Sloan Kettering Cancer Center, NewYork, NY

Purpose/Objective: NMR studies have indicated that choline (Cho) is elevated in rapidly growing tissues such as tumors. 1HMRS can be used to image the location of tumors within the prostate gland. One differentiates cancer from benign tissue byusing the ratio (Cho�Cr)/Cit [Cr � creatine; Cit � citrate] of the respective peaks in the MR spectrum. The ratios are calculatedon a spatial grid covering the prostate tissue. To incorporate this biological information, a voxel transformation (morphing)algorithm is designed for mapping points of interest from MRS images to ultrasound (US) images for target dose-escalationtreatment design. Robustness of the algorithm is tested, and plan quality and biological significance are evaluated.

Materials/Methods: Biological information was acquired via a GE Signa 1.5 Tesla MR Scanner. Radiofrequency excitationwas achieved by using the whole body birdcage resonator and the NMR signal was received using a 4 element phased arrayantenna combined with an expandable MRInnervu endorectal RF probe. Various images of the gland were taken. Mapping ofmetabolites over a 50-mm field of view was performed using chemical shift imaging to encode (6.25x6.25x6.25) mm3 voxels.Images and spectral data were then processed. Peak areas of Cho, Cr and Cit were calculated by numerical integration over thespectral ranges corresponding to each metabolite.

S371Proceedings of the 46th Annual ASTRO Meeting