Proceedings of the 32nd Annual ASTRO Meeting 193

terms of a “shadow field’ (i.e., the residual when the blocked field is subtracted from the open field), then the edge of a block as defined by the light field projection represents the Full-Width-at-Half-Maximum (FWHM) of the shadow field. The “maximum effective” block width can then be defined in terms of a percentage of the FWHM value. This data permits the physician to visualize the effective block size relative to that which is drawn on a simulator film. An additional important factor in designing mantle blocks is the thickness profile of underlying lung tissue. To determine this, the lung volume has been reconstructed from sequential CT scans and projected in the “beams eye” view using a color wash to indicate thickness. The results indicate the region where the blocks are most effective in protecting lung and where blocks can be modified to improve tumor dose without significantly affecting the dose-volume profile of the lungs.

132 RADIATION CARCINOGENESIS IN TRANSGENIC MOUSE MODELS: PROGRESS OF AN A.S.T.R.O. RESEARCH FELLOWSHIP

Eric H. Radany, M.D., Ph.D. and J. Michael Bishop, M.D. G.W. Hooper Foundation, U.C. San Francisco, San Francisco, CA

Ionizing radiations are capable of inducing neoplasia in a wide range of cell types; radiation carcinogenesis is thus of great interest to the molecular biologist, as well as being an important public health and clinical concern. Pairs of "activated" oncogenes have been shown to cooperate in the neoplastic transformation of normal diploid cells in several cases. This finding provides a rational approach to identifying the possible role of such oncogenes in radiation carcinogenesis: cells are first engineered to carry one activated oncogene. If ionizing radiation produces genetic changes (such as the activation of a second oncogene) that can cooperate with the first oncogene in transformation, enhanced radiation carcinogenesis should be observed in the cells. We are investigating an in vivo application of this strategy, using transgenic mouse lines that have been engineered to express an exogenous oncogene in specific tissues.

Work thus far has centered on creating transgenic mouse lines that will express oncogenes in the glial cells of the brain. These mice will be used to study the induction of gliomas by radiation, and they should also be useful in a variety of studies on the molecular biology of these tumors. The regulatory elements of several genes that express specifically in glial cells have been isolated by DNA cloning, and then linked to other genes (including oncogenes) to drive their expression in these cells. The recombinant DNA constr- ucts are now being tested for proper expression in glial cell cultures. Once this has been demonstrated, they will be introduced into the germline of mice by oocyte microinjection for the establishment of the transgenic mouse lines. These transgenic mice, as well and lymphoid tissue, will then be examined for possible from genetic cooperation with the transgenic oncogene. by molecular analysis of the tumors.

as similar ones that express oncogenes in breast increased radiation-induced tumorgenesis resulting The genetic lesion(s) responsible will be sought

133

REPAIR OF DNA DOUBLE STRAND BREAKS IN RADIATION RESISTANT CELLS OBTAINED BY TRANSFORMATION OF PRIMARY RAT EMBRYO CELLS WITH THE ONCOGENES H-RAS AND V-MYC

Marisa C. Weiss (1989-1990 ASTRO Research Fellow)(l), George Illiakis(3), Lorenz Metzger(3), Ruth J. Muschel(2), and W. Gillies McKenna(1)

(l)Department of Radiation Oncology; (2)Department of Pathology, University of Pennsylvania School of Medicine; Philadelphia, PA 19104 (3)Department of Radiation Oncology, Thomas Jefferson University School of Medicine: Philadelphia, PA 19107

Activation of the H-ras oncogene has been associated with resistance to ionizing radiation in a variety of cell lines. Recently it has also been shown that H-ras can act in a synergistic manner with the oncogene v-myc in conferring radiation resistance. These phenomena are of particular importance in Radiation Oncology since they may be partly associated with the known resistance to radiation of certain human tumors. Although the mechanism underlying this phenomenon is not presently known, it could be argued that the enhanced resistance to radiation, observed in cells transformed with ras, is conferred by an increase in DNA repair capacity. To test this possibility, repair of radiation-induced DNA damage was measured in two cell lines (3.7 and mycREC cells) derived from primary rat embryo cells (REC) by transformation with the oncogenes ras and myc. The 3.7 cell line was obtained by transforming REC cells with H-ras plus the cooperating oncogene myc (v-myc) and was found to be radioresistant. The mycREC cell line was obtained by immortalizing REC cells by retroviral transfection with pMVG/c-myc, and were used as controls. Among the DNA lesions induced by ionizing radiation, the double strand breaks (dsb) have often been implicated as the lesions responsible for cell killing. Induction and repair

194 Radiation Oncology, Biology, Physics October 1990,VOlume 19, Supplement 1

of DNA dsb were, therefore, studied in the above cell lines using a pulse field gel electrophoresis technique, the asymetric field inversion gel electrophoresis (AFIGE). Small variations were observed in the induction and repair of DNA dsb in these two cell lines that could be largely explained by differences in the distribution of cells in the various phases of the cell cycle. These results suggest that the radioresistance of 3.7 cells may not be mediated by alterations in their capacity to repair DNA dsb. It is hypothesized that alterations in repair and expression of potentially lethal damage underlies this phenomenon.

134

DIFFERENTIAL EXPRESSION OF THE C-JUN PROTO ONCOGENE DURING THE CELLULAR RESPONSE TO IONIZING RADIATION IS DE- MINISHED BY PROTEIN KINASE C INHIBITORS.

D.E. Hallahan, M.L. Sherman, M.A. Beckett, S. Virudachalam, D. Kufe, and R.R. Weichselbaum

Departments of Radiation and Cellular Oncology, University of Chicago, Chicago, IL and Laboratory Clinical Immunology, Dana-Farber Cancer Institute, Boston, MA

The molecular events which occur during the cellular response to ionizing radiation are poorly under- stood. Surviving clonogenic cells repair radiation damage, repopulate and produce cytokines after irradia- tion. RNA was analyzed from irradiated human normal tissues and tumor cell lines at varying time intervals to determine whether transcription factor gene induction occurs. Cells were grown to confluence and irra- diated with increasing doses of ionizing radiation (Z-20 Gy). RNA was isolated at 0.5, 1, 3 and 6 hours after irradiation. Northern blot analysis and hybridization with 32P labeled c-jun (transcription factor gene) plasmid were performed. c-jun demonstrated increased expression in a dose and time dependent manner for human tumor cell lines SQ-ZOB, RIT-3, STSAR-5 as well as normal tissue cell lines AG-1522 (fibroblast) and 293 (kidney). Differential expression of c-jun in response to irradiation is present since levels peak in tumor cells at 30 minutes but at 3 hours in normal tissues. Protein Kinase C (PKC) Inhibition resulted in deminished c-jun induction and enhancement of radiation cell killing. These data indicate that c-jun may be used as a model for immediate-early gene induction by ionizing radiation and may participate in the cascade of molecular events within surviving clonogenic cells. Furthermore, post transcriptional events (PKC) participate in the cellular response to radiation and inhibition of these events alters the radio- biological parameters.

135 DNA DAMAGE INDUCIBLE RESPONSES IN HUMAN CELLS

Edward N. Hughes+ and David Boothman*

Dept. of Kadiation Oncology, Univ. of PA+ & Univ. of Michigan*

The molecular events in the cellular response to DNA damage by ionizing radiation of human cells are not well understood. In order to investigate the biochemical mechanisms involved in potentially lethal damage repair (PLDR), a pleiotropic response activated in plateau phase human tumor cells by treatment with X-rays has been identified. Eight major X-ray induced proteins (XIP) of 126,000 to 275,000 MW were detected by resolving 35S-methionine labeled total cell proteins by use of 2-D gel electrophoresis. XIP expression was specific for ionizing radiation as heat shock, hypoxia, or alkylating agents failed to induce their synthesis. The expression of three classes of proteins was affected by X-irradiation. class I proteins, XIP145 and XIP269, were induced linearly with increasing x-ray doses. The rate of synthesis of Class II proteins, XIP126, XIP135, XIP138, XIP141, XIP147, and XIP275, increased with low X-ray doses, but plateaued at doses of 150-250 cGy. In contrast, the expression of Class III proteins, XIP47 and XIP254, decreased with increasing X-ray doses.

XIP269 expression was of particular interest: (1) it increased linearly by 25-fold with increasing radiation dose from 0.5 to 8.0 cGy; (2) it was distributed in a wide variety of human cells, but was completely absent in cells from patients with DNA-repair deficiencies to X-ray damage such as ataxia telangiectasia: (3) it strongly correlated with PLDR capacity: (4) it appeared to be cell-cycle regulated; and 15) it was inhibited specifically bycaffeine, an agent that induces premature mitosis and decreases cell viability following irradiation.

These data offer further evidence to support the hypothesis that XIP269 may be a regulatory protein required for the repair of X-ray induced damage. A possible role for this protein in cell cycle regulation and in DNA damage repair following X-irradiation will be discussed.