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//neomycin/oncogenes) FRANCISCO S. PARDO*t, ROBERT G. BRISTOWt, ALPHONSE TAGHIAN*, AUGUSTINUS ONG§, AND CARMIA BOREK§ *Department of Radiation , Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114; SPrincess Margaret Hospital, Toronto, ON Canada; and §Division of Radiation and 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 line, established from 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 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 The present set of experiments describe the in vitro (F.S.P., unpublished data).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 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 . 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 among radiation survival curves were mation studies, by injecting 5 x 104 and 1 x 106 cells into the derived using two-tailed tests of the respective MID. tail vein oflitter siblings from each ofthe animal strains used. Flow Cytometry. Because cell kinetics, in particular the A minimum of five animals were injected at each cell con- proportion of cells in S phase, can account for differences in centration for each clone. Lung colony formation was scored radiation sensitivity (18), flow cytometry was conducted to visually by one observer after lung tissue was fixed in picric determine the approximate cell-cycle distributions at time of acid for 24 hr. The lungs and liver of each animal were examined microscopically for evidence of metastases. Our prior experience with metastatic potential studies on murine cells transfected with oncogenes indicated that both spontaneous and experimentally induced metastases formed within 21 days of injection of 5 x 104 cells. For experiments with RECs either untransfected or transfected with a select- 0.1 able marker, we elected to delay animal sacrifice for 6 months after injection in those animals without tumors. For experi- ments with transfected or untransfected human glioma cells, our prior experience indicated that xenografts could form up to 13 months after injections of 1 x 107 cells (data not shown). I... We therefore elected to delay animal sacrifice up to 15 L.0.01 months for experiments with glial cells. Radiation Sensitivity. Radiation sensitivity experiments were conducted on exponentially growing cells 10-20 hours CD) after trypsin treatment and plating from stock cultures. Plating efficiency was determined for each line at the time of 0.0o ' c irradiation and flow cytometry. Multiplicity, the ratio of potential increase in cell number between time 0 (time of 0.001 plating) and time ofirradiation, was consistently less than 1.1. Initial experiments showed that there was no feeder-cell effect (9) on radiosensitivity or plating efficiency (data not shown). Cells were irradiated at room temperature, with a Table 2. Cell survival of glioblastoma cells after transfection and irradiation 3 5 7 9 DOSE Cell line Transfection SF2 MID (Gy) Hgl4 parent* None 0.44 2.43 FIG. 1. Radiation survival data for REC strains. The radiation Hgl4-1 pSV2neo 0.48 2.56 sensitivity of the untransfected (clones 1-6) and transfected (clones Hgl4-2 pCMVneo 0.45 2.49 7-10) clones is similar, as depicted in curve A. The radiation Hgl4-3 pSV2neo 0.40 2.41 sensitivities of clone 11 (curve B) and clone 12 (curve C) are Hgl4-4 pSV2neo 0.50 2.62 significantly different. Clones are identified as in Table 1. Individual pCMVneo 0.77 3.33 experiments are grouped by category ofsimilar radiosensitivity. Data Hgl4-5 are the mean and error bars represent the standard deviation from the *Includes subclones 1-7. mean. 10654 Genetics: Pardo et al. Proc. Natl. Acad. Sci. USA 88 (1991) Table 3. Flow cytometric celi-cycle analysis at time of irradiation of Hgl4 cells and RECs % of total cells Clone G, S G2/M REC-1 67.4 12.4 20.2 0.1 REC-2 62.0 18.0 19.0 REC-3 66.7 9.8 23.5 0 REC-4 58.5 16.4 25.1 0 REC-5 61.5 18.7 19.8 L.. REC-6 68.3 10.5 21.2 --III REC-7 70.5 9.5 20.0 0.01 REC-8 70.0 12.0 18.0 REC-9 61.5 19.0 19.5 REC-10 67.5 10.0 22.5 (I) REC-11 63.1 16.0 20.9 B REC-12 68.6 8.9 22.5 Hgl4 59.3 15.4 25.3 Hgl4-1 67.7 10.8 21.5 0.001 _- Hgl4-2 52.7 18.8 28.5 Hgl4-3 59.1 17.1 23.8 Hgl4-4 64.9 7.5 27.6 Hgl4-5 69.0 10.4 21.6 See Tables 1 and 2 for characterization of cells.

1 3 5 7 9 Table 2 and Fig. 2. One of the five neo-transfected Hgl4 DOSE (Gy) clones examined radiobiologically proved more resistant to ionizing radiation (P < 0.01) than the untransfected parental FIG. 2. Radiation survival data for the Hgl4 line (including seven line, parental subclones, or other clones from the same subclones of parent line) and neo-transfected clones. The radiation transfection experiment. The glial parent line and its trans- line and transfected clones (Hgl4-1- sensitivity of the parent Hgl4 fectant clones produced tumors in our immune-deficient mice Hgl4-4) is similar as seen in curve A. The radiation sensitivity of injection. There were no spontaneous or Hgl4-5 differs significantly as seen in curve B (clone identification as within 6 months of in Table 2). Individual experiments are grouped by category of experimentally induced metastases produced from injections similar radiosensitivity. Data are the mean and error bars represent of the untransfected glioma parent line or any of its neo- the standard deviation from the mean. transfected clones. Tumorigenicity of transfectant clones was similar to that ofthe untransfected parent line or parental irradiation. A total of 1 x 106 cells were harvested with cells subclones (data not shown). used for radiation survival plating. Individual flasks contain- Plating efficiencies were calculated at each experimental ing from 1 x 104 to 1 x 106 cells were processed and fixed session and ranged from 5 to 12% for the RECs and from within 2 hr of completion of irradiation on the matched 6-18% for the glioblastoma cells. There was no correlation parallel cohort of cells. Processing consisted oftrypsin treat- between plating efficiency and radiosensitivity within the ment, two washes in ice-cold phosphate-buffered saline, and series of experiments for a given clone of each category. storage on ice. Cells were then fixed in methanol/acetone, Flow cytometry data for RECs and Hgl4 cells showed that 1:1 (vol/vol), and stained in a solution ofpropidium iodide (10 from 7.5 to 19.0% of the cells were in S phase. There was no ,ug/ml) and RNase (1 mg/ml) in phosphate-buffered saline. correlation between increased numbers of cells in S phase Fluorescence-activated cell sorting analysis was conducted and relative radioresistance in vitro (Table 3). In addition, on 1 X 104 to 1 X 106 cells by using a Becton-Dickinson DNA indices from the same cytometric profiles did not show two-laser (488,633 nm) flow cytometer. Concomitant light any correlation between aneuploidy and radiosensitivity. scattering analysis was performed to note potential irregu- The use of two neo vectors containing simian virus 40 and larities in cellular size or cytoplasmic irregularities that might cytomegalovirus promoters in both REC and glioblastoma be indicative ofaltered cellular viability (19). The rare sample transfection experiments did not modify any of the parame- showing such irregularities was withdrawn from further con- ters studied. In addition, incubation of cells with calcium sideration. The data were modeled, using individual cell phosphate alone for 2-8 hr did not modify any of the fluorescence signal analysis, to approximate cell-cycle char- parameters studied. acteristics for the cells at the time of irradiation (20). DISCUSSION RESULTS The data presented above indicate that transfection ofnormal primary rat cells and human tumor cells with the neo plasmid, Two of six clones isolated from the transfected REC cultures and subsequent clonal selection, can give rise to cell popu- demonstrated increased resistance to ionizing radiation (P < lations that are significantly more resistant to ionizing radi- 0.05), compared to the untransfected RECs (Table 1 and Fig. ation than untransfected parent cells. This finding modifies 1). None ofthe REC strains produced tumors in our immune- the interpretation of oncogene transfection data that suggests deficient mice. In addition, there were no experimentally that the oncogenes alone alter radiosensitivity (1, 2). induced metastases in the lungs of animals injected with any Our studies indicate that there exists a significant amount of the REC strains. Transfection of RECs with neo did not of clonal heterogeneity in the radiation response of human alter the lifespan of the cells. Both untransfected and neo- and rodent cells transfected with a neo vector. One could transfected RECs senesced after 20-30 doublings. speculate that introduction of any exogenous sequence may Radiobiologic data for the human glioblastoma Hgl4 line, upset inherent stability, giving rise to variants from the host parental subclones, and its transfectant clones are found in line. Warnings about such effects evolve from studies where Genetics: Pardo et al. Proc. Natl. Acad. Sci. USA 88 (1991) 10655 calcium phosphate-mediated transfection of neo constructs showing that cells selected after oncogene transfection ex- were shown to induce and modify the metastatic behavior of pressed clonal heterogeneity not only with respect to tu- one murine tumor cell line (21). We performed all transfec- morigenicity but also with respect to metastatic potential. tions on the same parental cell population within two pas- Whether the propensity toward tumor formation is present sages. To allow for clonal heterogeneity in phenotypic only at low levels in the host background and would be expression, we performed radiation survival, flow cytomet- expressed only after the application of significant selective ric, and tumorigenicity assays in parallel on untransfected pressure in vitro/in vivo can only be surmised at this point. cells and transfected clones isolated from the same transfec- An individual clone must be considered a separate unit, tion experiment. potentially contributing to phenotypic heterogeneity. Such Our data emphasize the fact that neo transfection and the heterogeneity is well established both in vitro and in vivo, clonal selection process per se can have significant effects on where selective pressures in the milieu can induce hereditary cellular radiosensitivity. Such effects could depend upon a changes resulting in transformation, tumor formation, and variety offactors such as plasmid integration site, adjacent or metastasis (22, 24-26, 28). Interestingly, only recently have distant regulatory elements, and cellular metabolic status investigators emphasized phenotypic heterogeneity in the (including oxygenation and thiol levels). These diverse fac- response of cells to ionizing radiation (7, 27). Such hetero- tors can contribute to the overall "stability" of the host geneity has been shown in the expression of specific genome to transfection and clonal selection, thus complicat- in normal and oncogene-transformed RECs after irradiation. ing the scenario with respect to the role of oncogene trans- Heterogeneity in radiation responses correlated with a spe- fection in cellular radiosensitivity. cific subset ofgrowth-regulated cellular polypeptides includ- Transfection of neo vectors into RECs produced two ing cyclin and proliferating-cell nuclear antigen (27) and ras clones with a significant degree ofradiation resistance (Table p21 (F.S.P., unpublished data). 1). This neo effect was evident both in the clinically relevant We investigated the possibility that altered radiation sen- low-dose region (2 Gy) and in the higher-dose region (5-7 sitivity after neo transfection could be attributed to changes Gy). A similar resistance to ionizing radiation was seen with in cell kinetics at the time of irradiation. Transfection results the Hgl4 human glioma line transfected with neo vectors (SF2 are difficult to interpret because of the clonal heterogeneity = 0.77 vs. SF2 = 0.44 for the parent line) (Fig. 2). with respect to cell kinetics and regulation of expression. The fact that the neo effect occurs in both transfected These factors become more difficult to evaluate after expo- primary murine and tumorigenic human cells makes it a more sure to ionizing radiation. The issue of cell kinetics is general phenomenon. It emphasizes that adequate neo con- particularly relevant in this setting since cells are most trols must be included in each transfection experiment. In 10 resistant to the effects ofionizing radiation in late S phase and transfection experiments employing RECs, we found this relatively more sensitive in G2/M phases (18). Cell-density effect in two clones (Table 1). In 5 transfection experiments considerations at time of plating were addressed by using on human glioblastoma, one clone has shown this effect between 1 x 104 and 1 x 106 cells. This resulted in little or no (Table 2). One could speculate that indirect effects of trans- (-o5%) variation in either S phase or overall cell-cycle sta- fection must be considered with any direct mechanisms of tistics (data not shown). Flow cytometric determinations at oncogene action in modifying radiosensitivity. lower cell densities (<1 x 104 cells) were not conducted since We have emphasized MID and SF2 in assessing radiosen- these are close to the limits of resolution of the flow cytom- sitivity since we consider these to be the most discriminating eter. Flow cytometry was performed on untransfected and radiobiologic parameters with respect to low-dose in vitro transfected RECs and glioblastoma cells to investigate the radiosensitivity. SF2 and MID have been shown to be the best potential roles of cell-cycle effects and ploidy on radiation discriminators of radiosensitivity in vitro in human tumor sensitivity. Our flow cytometry data are presented in Table lines (10, 11) and cell lines (16). They have also shown a high 3. The results suggest that neither cell kinetic characteristics degree of correlation with the in vivo radioresponse of eight nor ploidy at the time of irradiation could explain the differ- murine tumors (14). MID is, by definition, identical to the ences in radiation survival parameters demonstrated in our area under the radiation survival curve obtained by integra- experiments. Transfectant clones and corresponding un- tion between zero and infinity in linear coordinates and in so transfected parental cells showed similar overall cell-cycle doing is more representative of the whole cell population characteristics. Cell kinetic perturbations induced by ioniz- rather than a fraction of it, thus minimizing cell survival ing radiation have not been addressed by us in these exper- fluctuations among various authors and experimental para- iments. DNA damage can induce arrest in the G2 phase of the digms (12). SF2, in particular, is being examined for its cell cycle, after DNA replication and before mitosis. Such a predictive value in that it represents cell survival after a delay may provide for DNA damage repair since cell division fraction ofthe amount of radiation commonly delivered in the in the presence of radiation-induced DNA damage can be clinic. The predictive value of these parameters is compli- lethal (29). A G2 delay after irradiation has been postulated as cated since in vitro cellular radiosensitivity is quite different a mechanism for the increased radiation resistance in RECs from the in vivo or clinical situation with respect to cellular transfected with myc and activated ras oncogenes (30). Such reoxygenation, repair, reassortment, and repopulation. cell-cycle mechanism(s) could also contribute to clonal het- There is as yet not enough published data to conclusively erogeneity in radiation sensitivity after neo transfection and demonstrate the predictive value of SF2, though there are clonal selection. indeed promising preliminary results (17). Our results indicate that transfection with the neomycin- Since selective pressures have been shown to encourage selectable marker and subsequent clonal selection can mod- neoplastic transformation (22, 28), we investigated whether ulate the radiation sensitivity of both murine and human cells neo transfection modifies the neoplastic potential of the cells in vitro without significantly altering cellular kinetics at the and whether such changes might correlate with altered radi- time of irradiation, tumorigenicity, or metastatic potential. osensitivity. Tumorigenicity was not modified by neo trans- The mechanisms underlying these effects warrant further fection. Only the parent glial line and its five transfectant investigation, since the modulation of inherent radiosensitiv- clones were tumorigenic in our immunodeficient mice. Tu- ity is emphasized as an important factor in clinical radiother- morigenicity of transfectants approximated that of the un- apeutic response (10-12, 14-17). transfected parental line. None of the REC strains showed evidence of tumor formation or experimental metastases. We thank Drs. Herman D. Suit, Emmett Schmidt, and Kathyrn D. This is distinct from other data from our laboratories (7) Held for helpful suggestions and Ms. Pam Edwards, Ms. Marlene 10656 Genetics: Pardo et al. Proc. Natl. Acad. Sci. USA 88 (1991)

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