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Downloaded at Google Indexer on July 22, 2021 Downloaded by guest on September 29, 2021 Proc. Nati. Acad. Sci. USA Vol. 88, pp. 10652-10656, December 1991 Genetics Role of transfection and clonal selection in mediating radioresistance (gene transfer/radiosensitivity/neomycin/oncogenes) FRANCISCO S. PARDO*t, ROBERT G. BRISTOWt, ALPHONSE TAGHIAN*, AUGUSTINUS ONG§, AND CARMIA BOREK§ *Department of Radiation Oncology, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114; SPrincess Margaret Hospital, Toronto, ON Canada; and §Division of Radiation and Cancer Biology, Department of Radiation Oncology, Tufts University School of Medicine/New England Medical Center, Boston, MA 02111 Communicated by Harry Rubin, August 12, 1991 ABSTRACT Transfected oncogenes have been reported to is a radiobiologically well-characterized early-passage glio- increase the radioresistance of rodent cells. Whether trans- blastoma cell line, established from human operative material, fected nononcogenic DNA sequences and subsequent clonal at Massachusetts General Hospital. Immunoperoxidase data selection can result in radioresistant cell populations is un- for glial fibrillary acidic protein substantiates its glial origin known. The present set of experiments describe the in vitro (F.S.P., unpublished data). Subclones ofthe parental glioblas- radiosensitivity and tumorigenicity of selected clones of pri- toma cells were established from the initial tumor biopsy. All mary rat embryo cells and human glioblastoma cells, after cultures were passaged and maintained in Dulbecco's modi- transfection with a neomycin-resistance marker (pSV2neo or fied Eagle's medium (DMEM, Sigma) fortified with 10o pCMVneo) and clonal selection. Radiobiological data compar- (vol/vol) fetal calf serum (Rehatuin, St. Louis). Cells were ing the surviving fraction at 2 Gy (SF2) and the mean inacti- incubated in a humidified atmosphere of 5% C02/95% air at vation dose show the induction of radioresistance in two rat 37°C. Cells were passaged at late logarithmic phase to perpet- embryo cell clones and one glioblastoma clone, as compared to uate stock cultures (4). untransfected cells. Wild-type and transfectant clones were Transfection. RECs were transfected between passages 2 injected into three strains ofimmune-deficient mice (scid, NIH, and 4. RECs and Hgl4 cells were transfected by the standard and nu/nu) to assay for tumorigenicity and metastatic poten- calcium phosphate technique as described by Ausubel et al. tial. Only the glioblastoma parent line and its transfectant (5). The two neo expression vector constructs used were clones were tumorigenic. None of the cells produced sponta- pSV2neo (SV2 promoter, gift of R. Bernards, Massachusetts neous or experimentally induced metastases. Flow cytometric General Hospital Cancer Center) and pCMVneo (CMV pro- analyses indicated that the induction of radioresistance could moter, gift of E. Schmidt, Massachusetts General Hospital not be attributed to changes in cell kinetics at the time of Cancer Center). Neo vector restriction maps were confirmed irradiation. Our results show that transfection of a neomycin- prior to transfection with appropriate restriction enzymes. resistance marker and clonal selection can impart radioresis- Plasmids were not linearized prior to transfection. tance on both normal and tumor cells. The work also indicates For calcium phosphate-mediated transfections, 30 jig of that altered radiation sensitivity does not necessarily correlate the neo construct was used per 10-cm plate. Two to 8 hr after with changes in cell-cycle kinetics at the time of irradiation, calcium phosphate application, cells were treated with 15% tumorigenicity, or altered metastatic potential. Our findings (vol/vol) glycerol in DMEM for 3 min. The selection medium have critical implications for transfection studies investigating for neo-transfected cells was DMEM supplemented with determinants of cellular radiosensitivity. G418 (Sigma). The final G418 concentrations chosen were 200 ,ug/ml for selecting RECs and 500 ,tg/ml for selecting Recent developments in molecular biology have stimulated Hgl4 cells. These levels of G418 proved toxic to the untrans- interest in the possibility that certain oncogenes might play a fected RECs and Hgl4 cells, respectively. Transfectant role in determining cellular responses to ionizing radiation. clones were isolated after 2-3 weeks in selection medium. The interest emanates from a belief that tumor development Both REC and Hgl4 transfectants were continuously main- and metastatic potential might correlate with radioresistance tained in the G418 selective medium. (1). Several studies have indicated that transfection with Tumorigenicity. To test for tumor formation, 0.5 ml con- mutated ras genes can increase the resistance of NIH 3T3 taining 1 x 105, 1 x 106, or 1 x 107 cells was injected into the cells (2) or rat embryo cells (RECs) (1) to ionizing radiation. axillary pouch of three strains of immune-deficient mice The present study was undertaken to determine whether (NIH III, nu/nu, scid), housed in the Department of Radia- transfection with the neomycin-resistance marker (neo) gene, tion Oncology Animal Facility at Massachusetts General commonly used for selection in transfection studies, alone Hospital. A minimum of five animals were injected at each can modify the cellular radiosensitivity, tumorigenicity, and cell concentration for each clone. More than one murine metastatic potential of transfected cells. strain was used since prior work suggested that subtle residual immunologic differences exist among strains (6). MATERIALS AND METHODS Animals were not further immunosuppressed with radiation before injection of untransfected or transfected cells. For the Cells. Primary embryo cells (RECs) were prepared from 7- tumorigenic glioma line, tumorigenicity was measured as the to 14-day Sprague-Dawley rat embryos (Charles River Breed- time elapsed between injection and attainment of a tumor ing Laboratories) by using established methods (3). The cell strains survived for 20-30 doublings before senescence. Hgl4 Abbreviations: REC, rat embryo cell; MID, mean inactivation dose; neo, neomycin-resistance marker; SF, surviving fraction. The publication costs of this article were defrayed in part by page charge tTo whom requests should be addressed at: Massachusetts General payment. This article must therefore be hereby marked "advertisement" Hospital, Department of Radiation Oncology, Cox Cancer Cen- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ter-3, Boston, MA 02114. 10652 Genetics: Pardo et al. Proc. Natl. Acad. Sci. USA 88 (1991) 10653 Table 1. Cell survival of RECs after transfection and irradiation 250-kV-peak x-ray unit 0.44-mm Cu half value layer at a dose Cell strain(s) Transfection SF2 MID rate of 1.54 Gy/min. Cultures were incubated from 10 to 14 days (REC strains) or 21 days (Hgl4 line) for subsequent REC-1,2,3 None 0.38 1.53 colony formation. Cell cultures were then fixed with 100% REC-4,5,6 None 0.39 1.58 methanol, stained with methylene blue, and scored for col- REC-7,8 pSV2neo 0.42 1.67 onies of 50 cells or more. For each transfection experiment, REC-9,10 pCMVneo 0.47 1.71 individual clones were analyzed (to address issues of clonal REC-11 pSV2neo 0.59 2.05 heterogeneity). For the parental and transfected REC strains, REC-12 pSV2neo 0.62 2.60 two or three sets of survival data were obtained for each SF2, surviving fraction after x-irradiation with 2 Gy fitted to a clonal population prior to senescence. For the glioma line and linear quadratic model (see text); MID, mean inactivation dose its neo transfectants, a minimum ofthree sets of survival data representing total area under radiation survival curve (12). were obtained for each cell line. All cell survival determina- with a 1.2-cm maximum diameter, at which time the tumors tions were conducted in triplicate resulting in a minimum of were harvested. six determinations at each dose level for the RECs and a Our prior experience with injecting murine cells trans- minimum of nine determinations at each dose level for the fected with oncogenes indicated that most tumors form glioblastoma cells. within 1 month ofinjection (7). For experiments with primary Cell survival measurements were fitted to a linear qua- RECs either untransfected or transfected with the selectable dratic mathematical model (10, 11). The results are presented marker, we elected to delay animal sacrifice for 6 months in Tables 1 and 2 and Figs. 1 and 2. The experimental data are after injection in those animals without tumors. For experi- arranged to compare relative radiosensitivity while simulta- ments with transfected or untransfected human glioma cells, neously addressing possible clonal heterogeneity after trans- we delayed sacrifice for 15 months since our earlier work with fection. We have emphasized the SF2 and the MID, which establishing human xenografts indicated that tumor develop- takes into account the total area under the radiation cell ment may take longer than a year (6, 8). survival curve. These are considered, by many authors, to be Metastatic Potential. For tumorigenicity studies, lungs and the most discriminating radiobiologic parameters oflow-dose liver were harvested from each animal at necropsy to assay radiosensitivity in vitro (10, 12-17). SF2, in particular, may be for spontaneous metastases. Assays for experimental meta- predictive of clinical radiotherapeutic response (15, 17). P static potential were performed in parallel with tumor for- values for comparison
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