INT . J . RADIAT . BIOL ., 1990, VOL . 58, NO . 3, 509-517
Response of cultured human airway epithelial cells to X-rays and energetic ac-particles
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T . C . YANGt, D . C . GRUENERT$, W . R . HOLLEYt and S . B . CURTISt tCell and Molecular Biology Division, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720, USA Cardiovascular Research Institute and Cancer Research Institute, University of California-San Francisco, San Francisco, CA 94143, USA
(Received 30 November 1989 ; revision received 14 March 1990 ; accepted 29 March 1990) Radon and its progeny, which emit a-particles during decay, may play an important role in inducing human lung cancer . To gain a better understanding of the biological effects of a-particles in human lung we studied the response of cultured human airway epithelial cells to X-rays and monoenergetic helium ions . Our experimental results indicated that the radiation response of primary cultures was similar to that for airway epithelial cells that were transformed with a plasmid containing an origin-defective SV40 virus . The RBE for cell inactivation determined by the ratio of Do for X-rays to that for 8 MeV helium ions was 1 . 8-2 . 2 . The cross-section for helium ions, calculated from the Do value, was about 24 µm 2 for cells of the primary culture . This cross-section is significantly smaller than the average geometric nuclear area (- 180µm 2 ), suggesting that an average of 7 . 5 a-particles (8 MeV helium ions) per cell nucleus are needed to induce a lethal lesion .
1.
Introduction Epidemiological studies have shown that radon and its progeny may play an important role in the lung cancer problem of uranium miners (NCRP 1984) . Recent findings showing high levels of radon in many homes have caused great concern as to the potential risk of radon, and have increased interest in the biological effects of a-particles (Stranden 1980) . Alpha-particles released during the decay of radon progeny are implicated as an important factor in lung cancer induction (NCRP 1984) . Because 85-90°,10 of all cancers are epithelial in origin, a better understanding of the biological effects of a-particles in human lung will result from the study of the radiation response of human airway epithelial cells directly . Recent developments in the culture of human airway epithelial cells have now made such studies possible (Lechner and LaVeck 1985, Gruenert et al. 1988) . We initiated studies on the lethal effects of X-rays and energetic a-particles in human bronchial and tracheal epithelial cells in order to fill an important gap in our knowledge of the radiation response of human airway epithelial cells . This paper reports the results obtained with primary cultures and airway epithelial cells that were transformed with an origin-defective simian virus 40 (SV40) containing plasmid (Gruenert et al . 1988) . 2.
Materials and methods
2 .1 . Cell culture All cultures were human in origin and derived from human trachea or bronchus following autopsy or termination of pregnancy . The isolation and culture of human 0020-7616/90 $3 .00 .~ ; 1990 Taylor & Francis Ltd
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airway epithelial cells has been reported in detail elsewhere (Gruenert et al. 1990) . Briefly, tissue was processed within 24 h of removal . All airway tissue was washed with cold (4°C) phosphate-buffered saline (PBS) containing penicillin (100 U/ml), streptomycin (100µg/ml) and amphotericin B (50µg/ml) . Tracheal and bronchial epithelial cultures were initiated for tissue explants . Explant cultures were established by cutting tissue into 2 mm square pieces and then placing them on tissue culture dishes coated with fibronectin (Collaborative Research Inc .), vitrogen 100 (Collagen Corporation, Palo Alto, CA) and bovine serum albumin (Biofluids, Rockville, MD) . The dishes were coated with a solution containing 1 mg fibronectin, l ml vitrogen 100, 10 ml of 10% bovine serum albumin stock solution and 100 ml basal growth medium (FN-V-BSA) . The coating solution was incubated in the dishes at 37 ° C for 3 h . After incubation the coating solution was removed, and the dishes were dried at room temperature before use . Explants were covered with dialysed serum to facilitate attachment and incubated for 24 h at 37°C under 5 % CO 2 in air . Serum was then diluted with serumfree medium . The serum-free medium is a modified form of LHC-9 medium (Lechner and LaVeck 1985) containing glutamine and no retinoic acid and is designated MLHC-8e (Gruenert et al . 1990) . LHC-8 (Biofluids) is supplemented with epinephrine (0 . 5 µg/ml) . This LHC-8e medium was further modified (Gruenert et al . 1990) . The Stock 4 and trace element components of the medium were increased by 20% . The final concentration of glutamine was 4mM . Since blood-derived serum contains tumour growth factor-/3 (TGF-fl), an agent that induces squamous cell differentiation, and because the growth of contaminating fibroblasts is inhibited in serum-free medium (Lechner and LaVeck 1985), cells were put into serum-free medium as soon as possible after isolation (about 48 h after explanting) . After epithelial outgrowth appeared, explants were transferred to other dishes to generate additional epithelial cultures . Explant cultures are often mixed populations of epithelial cells and fibroblasts . Generation of cultures that are exclusively epithelial was achieved by selective trypsinization (Noyes et al . 1980, Gruenert et al . 1990) . The mixed cultures were washed twice with a HEPES buffered saline (HBS) (18 . 3 mm HEPES, 121 mm NaCl, 2-7 mm KCI, 9-4 mm glucose, 7 . 2 mm Na 2 HPO 4 -7H 2 0), phenol red (0 . 5%), 0 . 25 ml/l ; (pH 7 . 45) (Lechner and LaVeck 1985 ; Gruenert et al . 1990) . The cells were then incubated at 37°C in a trypsinization cocktail containing 0 . 02% EGTA, 1 % polyvinylpyrrolidine (PVP), and 0 . 02% trypsin, 0 .0016% EDTA in Puck's saline A and 80 mg/l glucose . Fibroblasts were detached by rinsing once with the trypsinization solution followed by two additional rinses with HBS . Attached cells were incubated at 37°C overnight in the presence of MLHC-8e medium under 5% CO 2 in air and assessed the following day for further fibroblastic contamination . Selective trypsinization was repeated within 48 h of the initial trypsinization if there was still morphological evidence of fibroblasts . Cells were subcultured using the same trypsinization solution as described above when they reached 80-90% confluence . Cultures were rinsed twice with HBS, and then incubated at 37°C in the trypsinization solution . The cells were then pelleted and resuspended in MLHC-8e medium . New cultures were established at a split ratio of between 1 : 2 and 1 :4 on tissue culture plastic coated with FN-V-BSA as described above . Growth medium was routinely replaced with fresh LHC-8e every other day . Transformed tracheal epithelial cells were obtained by transfecting the primary
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cultures with a plasmid containing an SV40 virus defective in the origin of replication (pSVori-) (Gruenert 1987, Gruenert et al . 1988) . The post-crisis 9HTEo line has grown well in Eagle's MEM supplemented with 10% fetal calf serum, glutamine and antibiotics . The 9HTEo cells are immortal and are no longer contact-inhibited . They also exhibit anchorage-independent growth by forming colonies in soft agar . No tumorigenicity was indicated after injecting 10 6 cells into athymic nude mice (unpublished observations) . The pre-crisis cells used for survival studies were obtained a few passages after pSVori- transfection . These cells were grown and subcultured in the same way as cultures of non-transformed epithelial cells . The epithelial origin of the cells was determined immunocytochemically . All cultures were assayed for the presence of keratin, an epithelial-specific marker (Gruenert et al . 1988, 1990) . The presence of epithelial-specific markers such as keratin, tight junction formation, microvilli containing actin-like filaments, and ion transport has been previously verified for cultures of both transformed and nontransformed epithelial cells used in these studies (Gruenert et al. 1988, 1990, respectively) . 2 .2 . Irradiation The Super HILAC at LBL provided the monoenergetic helium beams for all the radiation studies . Routine beam monitoring and exposure dosimetry were done by using a thin-walled parallel plate ionization chamber immediately downstream of the beam exit window and adjacent to the sample exposure position . The calibration of the ion chamber was made by means of a beam total-charge collector (Faraday cup) located at the sample exposure position . The energy of the helium beam was determined with a silicon detector . The uniformity of the beam within the irradiated sample area and the accuracy of dose was within 5% . About 10-12 h before irradiation, exponentially growing stock cells were dissociated with a trypsinization solution containing PVP, EGTA, trypsin and EDTA (Gruenert et al . 1988) . Cells were plated at a density giving about 50 colonies per dish for each treatment dose . These dishes were checked for multiplicity shortly before radiation exposure . In these studies very few doublets were found, possibly due to the long doubling time (,> 36 h) . Cell growth medium was removed from the dish just before irradiation . In order to minimize the problem of cell drying the dose rate was carefully adjusted so that the irradiation time for all doses used was less than 20 s . Immediately after irradiation, fresh medium was added to the cells, and the dish was then incubated for 2-3 weeks at 37°C to allow colony formation . At the end of the incubation period, cells were fixed and stained, and colonies containing more than 50 cells were counted as survivors . The present study thus used only the standard clonogenic assay . The possible induction by radiation of lethal mutations in the progenies of survivors, as found by other investigators (Seymour et al . 1987, Gorgojo and Little 1989), were not studied . For radiation experiments with primary cultures and pre-crisis cells, fibronectin-coated dishes and feeder cells were used . About 5 x 10 4 mouse fibroblasts, given 50 Gy of X-rays, were seeded into each dish as the feeder cells . Four dishes were used for each radiation dose, and six dishes for the control . X-ray experiments were performed with a 250 kVp Philips X-ray machine, operated at 225 kVp, 15 mA, with 0 . 35 Cu filter and a dose rate of 85 cGy/min . Tissue culture dishes (60 mm) were irradiated on a rotating table, and the uniform-
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ity of dose over the whole area of dish was better than 3%, as measured by a Victoreen ionization chamber . 2 .3 . Data analysis Survival data obtained from these experiments were fitted by a computer program, using the least-squares method, to determine the a and /3 values for the linear-quadratic model and n and D o for the multi-hit and single-target model . The standard errors for the survival data were calculated from the formula SE=(n) t12 /N, where n is the total number of colonies counted and N is the number of plates used . The cross-section was calculated using the following equation : a (µm2)=[0 .16 x LET (keV/um)]/Do (Gy) where a is the cross-section and D o is obtained from the final slope of the survival curve (Curtis 1988) . 3.
Results Figure 1 shows the results for the immortal 9HTEo cells irradiated by X-rays and 8 MeV helium ions . The X-ray survival curve showed a significant shoulder (extrapolation number n of 3 . 2) and a D o about 120cGy . The survival curve for
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Figure 1 . Dose-response curves for survival of immortal human airway epithelial cells, post-crisis, P3-83 (9HTEo) irradiated by X-rays and 8 MeV helium ions . The average plating efficiency was 30% .
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helium ions appeared to be close to exponential with a D o about 59 cGy . The RBE determined by the ratio of D o for X-rays to that for helium ions was 2-0 (Table 1) . The RBE determined at 10% survival level, however, was slightly higher, about 2-4 . The survival curves for primary cultures are shown in Figure 2 . For X-rays the n and D o were 1-86 and 128 cGy, respectively . The extrapolation number and D o for helium ions were 1-67 and 57-5 cGy, respectively . The RBE determined by the final slope was 2-2, similar to that measured at 10% survival level (2 . 3) . The X-ray survival curve for pre-crisis cells indicates an n of 2 . 35 and a Do of 142 cGy (Figure 3) . The n and Do for helium ions was 1 . 55 and 79 cGy, respectively . The RBE was 1-79 as determined by the final slopes and 2 .1 at 10% survival level . The a-values for all survival curves show a 2-3-fold increase for cells irradiated by helium ions, as compared with those irradiated by X-rays (Table 1) . For primary cultures, for example, the a was 435 x 10 -3 cGy - ' and 11-17x 10 -3 cGy - ' for X-rays and helium ions, respectively . The fl-value of X-rays was very similar for -2 . primary, pre-crisis and post-crisis cells, about 4-6 x 10 -6 cGy The #-value for helium ions showed a large standard deviation, suggesting that the survival curves, especially for primary and post-crisis cells, might be fitted equally well by a pure exponential function . For determining the probability of interaction between the helium ions and the target in the cell we have calculated the cross-sections . The cross-sections for helium ions, calculated from the Do values, indicated no significant difference between primary culture and post-crisis cells, at about 24 µm 2 (Table 1) . The crosssection of pre-crisis cells was about 17-5µm 2 , somewhat smaller than that for primary culture and post-crisis cells . 4.
Discussion Progress in quantitatively understanding the radiation response of human epithelial cells has been limited by the difficulty of growing these cells in vitro . Table 1 . Inactivation of human airway epithelial cells by helium ions and X-rays
Cells Primary
Number Alpha Beta of experi(x 10 - 3 (x 10 -6 2) ments Radiation cGy - ') cGy_ 2
X-rays He
Precrisis
2
X-rays He
Postcrisis
3
X-rays He
n
o (cGy) D
4 . 35 ±0-58 11-17 +2 . 98
4-66 +1 . 61 18-06 +16 . 23
1 . 86 +0 . 38 1-67 +0 . 47
128-5 +3 . 1 57 . 7 +5 . 2
2-96 ±0 . 32 7 . 25 _ 0 . 84 + 2-65 +0-51 12-58 ±2 . 38
4-43 ±0 . 57 16-1 +448 6-51 +1-04 10-34 ±12 . 87
2-35 ±0 . 42 1-55 _ 0 . 22 + 3 . 21 +0-62 1 . 53 ±0 .43
141-8 ±7 . 1 79-4 +4 .8 119-9 +5-7 59-2 ±5 . 5
* Dox is D o for X-rays, and Do„, for helium ions .
RBE (Do , / Do „.)*
Crosssection (µm 2 )
1-0 2-23 +0 . 05
24-2 +2 . 0
1 .0 1-79 +0 . 08 1 .0
17-5 +1 . 0
2 . 03 ±0 . 09
23-5 ±2 . 0
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Figure 2 . Survival curves for primary culture of human airway epithelial cells irradiated by X-rays and 8 MeV helium particles . The average plating efficiency was 15°c . Recent advances in epithelial culture have facilitated assessment of the radiosensitivity of several types of human epithelial cells . Human mammary (Gathers and Gould 1983, Yang et al. 1983, 1989), thyroid (Miller et al. 1987, Seymour and Mothersill 1987), epidermis (Dover and Potten 1983, Kasid et al . 1987) and umbilical cord vein (Hei et al . 1987, Hall et al . 1988) epithelial cells have all been studied using clonogenic assays . Our results with human airway epithelial cells were very similar to those reported for epidermal keratinocytes (Kasid et al . 1987), i .e . no significant difference in radiosensitivity between primary cultures and established cells . There is, however, a significant difference in D o between airway epithelial cells (D o = 120-130 cGy) and epidermal keratinocytes (D o =224-243 cGy) . This difference is not totally unexpected since different D o values have been observed for thyroid and mammary gland epithelial cells . Whether this difference in radiosensitivity between cells from different tissues is due to differences in repair mechanisms or other epigenetic factors, e .g . extracellular matrix, is unknown at present . Investigators at several laboratories have determined the RBE values for a-particles with various energies in various cell systems . For human fibroblasts, for example, RBE values ranging from 1 . 4 to 4 . 0 have been reported for accelerated helium ions with LET from 20 to 90keV/µm, respectively (Cox et al . 1977) . The lethal effect of a-particles with LET of 110-200keV/µm was studied with human T-1 cells, and an RBE of 8 was found at low doses of a-particles with
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0
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515
-RBE ; 2 .1 10
0 5 tt
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Figure 3 . Survival curves for pre-crisis human airway epithelial cells irradiated by X-rays and 8 MeV helium ions . The average plating efficiency was 21 % .
an LET of 110 keV/µm (Barendsen 1964) . Our studies with 8 MeV helium ions (- 87keV/µm) showed that the RBE for primary and established human tracheal epithelial cells was 2 . 2-2 . 4 and is in the range of RBE values reported for other human cell systems . Previous studies have suggested that cells in the more advanced stages of neoplastic progression have increased sensitivity to low-LET radiation (Gantt et al . 1987, Sanford et al. 1987) . Our observations of cell survival show no difference in X-ray or a-particle sensitivity between cells in progressive stages of transformation or non-transformed cells . These results are supported by those found by Bender et al . (1988) . Using the experimental data, we calculated the cross-sections for cell inactivation . Use of the D o of the final slope of the survival curve to calculate the crosssection is valid for an exponential curve with no change of slope . The cross-section calculated from the D o of a shouldered survival curve, in general, will be larger than that from the initial slope which gives the true cross-section value . Because the survival curve of helium ions has only a very small shoulder, i .e . is nearly exponential, the calculated cross-section value should not differ significantly from the true value .
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The cross-section for human airway epithelial cells, determined from the helium ion survival curves, was about 24pm 2 , which is significantly smaller than their average geometric nuclear area (-180 µm 2 ) . These results suggest that an average of 7 . 5 helium ions per cell nucleus are needed to induce a lethal lesion . Therefore, some 85% of the helium ions traversing a cell nucleus do not result in lethality . A single traversal of an a-particle in the cell nucleus, therefore, could cause some non-lethal effects, such as mutations and/or chromosomal aberrations . At present it is unknown whether a single a-particle traversing the cell nucleus is sufficient to induce neoplastic transformation . Understanding the carcinogenic
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effect of a-particles in human airway epithelial cells, especially at low doses, is particularly important for assessing the potential cancer risk of radon exposure in the domestic environment . Neoplastic cell transformation experiments with human airway epithelial cells are now under way in our laboratory . Acknowledgements The expert typing and editing of Tennessee Gock is appreciated . We also thank Laurie Craise and Sujay Dutta for their technical assistance . This research is supported by the Office of Health and Environmental Research, US Department of Energy, under Contract DE-AC03-76SF00098, and NIH Grant DK39619 (DCG) . References BARENDSEN, G . W., 1964, Impairment of the proliferative capacity of human cells in culture by alpha particles with differing linear-energy transfer . International Journal of Radiation Biology, 8, 453-466 . BENDER, M . A ., VIOLA, M . V ., FIORE, J ., THOMPSON, M . H . and LEONARD, R . C ., 1988, Normal G 2 chromosomal radiosensitivity and cell survival in cancer familial syndrome . Cancer Research, 48, 2579-2584 . CATHERS, L . E . and GOULD, M . N ., 1983, Human mammary cell survival following ionizing radiation . International Journal of Radiation Biology, 44, 1-16 . Cox, R ., THACKER, J ., GOODHEAD, D . T . and MUNSON, R . J ., 1977, Mutation and inactivation of mammalian cells by various ionizing radiations . Nature, 267, 425-427 . CURTIS, S ., 1988, Radiation physics related to biology . Terrestrial Space Radiation and Its Biological Effects, edited by P . D . McCormack, C . E . Swenberg and H . Bucker (Plenum, New York), pp . 301-313 . DOVER, R . and POTTEN, C . S ., 1983, Radiosensitivity of normal human epidermal cells in culture . International Journal of Radiation Biology, 6, 681-685 . GANTT, R ., SANFORD, K . K ., PARSHAD, R ., PRICE, F . M ., PETERSON, W . D ., JR and RIHM, J . S ., 1987, Enhanced G 2 chromatic radiosensitivity at an early stage in the neoplastic transformation of human epidermal keratinocytes in culture . Cancer Research, 47, 1390-1397 . GoRGOJo, L . and LITTLE, J . B ., 1989, Expression of lethal mutations in progeny of irradiated mammalian cells . International Journal of Radiation Biology, 55, 619-630 . GRUENERT, D . C ., 1987, Differentiated properties of human epithelial cells transformed in vitro . Biotechniques, 5, 740-748 . GRUENERT, D . C ., BASBAUM, C . B ., WELSH, M . K ., LI, M ., FINKBEINER, W . E . and NADEL, J . A ., 1988, Characterization of human tracheal epithelial cells transformed by an origin-defective SV40 . Proceedings of National Academy of Sciences, USA, 85, 5951-5955 . GRUENERT, D . C ., BASBAUM, C . B . and WIDDICOMBE, J . M ., 1990, Long term culture of normal and cystic fibrosis epithelial cells grown in serum free medium . In Vitro, 96, 411-418 . HALL, E . J ., MARCHESE, M ., HEi, T . K . and ZAIDER, M ., 1988, Radiation response characteristics of human cells in vitro . Radiation Research, 114, 415-424 .
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HEI, T . K ., MARCHESE, M . J . and HALL, E . J ., 1987, Radiosensitivity and sublethal damage repair in human umbilical cord vein endothelial cells . International Journal of Radiation Oncology, Biology, Physics, 13, 879-884.
KASID, U . N ., RHIM, J . and DRITSCHILO, A ., 1987, Human epidermal keratinocytes retain radiation resistance following in vitro immortalization and malignant transformation . Radiation Research, 111, 565-571 .
LECHNER, J . F . and LAVECK, M . A ., 1985, A serum-free method for culturing normal human bronchial epithelial cells at clonal density . Journal of Tissue Culture Methods, 9,
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43-48 .
MILLER, R . C ., HIRAOKA, T ., KOPECKY, K . J ., NAKAMURA, N ., JONES, M . P ., ITO, T . and CLIFTON, K . H ., 1987, Sensitivity to radiation of human normal, hyperthyroid, and neoplastic thyroid epithelial cells in primary culture . Radiation Research, 111, 81-91 . NCRP, 1984, Evaluation of Occupation and Environmental Exposures to Radon and Radon Daughters in the United States . NCRP report No . 78 (National Council on Radiation Protection and Measurement) . NOYES, I ., MILL, G . and CUNNINGHAM, C ., 1980, Establishment of proliferating human epithelial cells in vitro from cell suspension of neonatal foreskin . Tissue Culture Association Manual,
5, 1173-1175 .
SANFORD, K . K ., PARSHAD, R ., GANTT, R ., TARON, T . E ., JOHNS, G . M . and PRICE, F . M ., 1987, Factors affecting and significance of G Z chromatin radiosensitivity in predisposition in cancer . International Journal of Radiation Biology, 51, 381-391 . SEYMOUR, C . B . and MOTHERSILL, C ., 1987, Long-term effects of gamma irradiation on cultured human thyroid cells . International Journal of Radiation Biology, 51, 381-391 . SEYMOUR, C . B ., MOTHERSILL, C . and ALPER, T., 1987, High yields of lethal mutations in somatic mammalian cells that survive ionizing radiation . International Journal of Radiation Biology, 50, 167-179 .
STRANDEN, E ., 1980, Radon in dwellings and lung cancer-a discussion . Health Physics, 38, 301-306 .
YANG, T . C ., STAMPFER, M . R . and SMITH, H . S ., 1983, Response of cultured normal human mammary epithelial cells to X-rays . Radiation Research, 96, 476-485 . YANG, T . C ., STAMPFER, M . R . and TOBIAS, C . A ., 1989, Radiation studies on sensitivity and repair of human mammary epithelial cells . International Journal of Radiation Biology, 56,605-609 .
Response of cultured human airway epithelial cells to X-rays and energetic ac-particles
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T . C . YANGt, D . C . GRUENERT$, W . R . HOLLEYt and S . B . CURTISt tCell and Molecular Biology Division, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720, USA Cardiovascular Research Institute and Cancer Research Institute, University of California-San Francisco, San Francisco, CA 94143, USA
(Received 30 November 1989 ; revision received 14 March 1990 ; accepted 29 March 1990) Radon and its progeny, which emit a-particles during decay, may play an important role in inducing human lung cancer . To gain a better understanding of the biological effects of a-particles in human lung we studied the response of cultured human airway epithelial cells to X-rays and monoenergetic helium ions . Our experimental results indicated that the radiation response of primary cultures was similar to that for airway epithelial cells that were transformed with a plasmid containing an origin-defective SV40 virus . The RBE for cell inactivation determined by the ratio of Do for X-rays to that for 8 MeV helium ions was 1 . 8-2 . 2 . The cross-section for helium ions, calculated from the Do value, was about 24 µm 2 for cells of the primary culture . This cross-section is significantly smaller than the average geometric nuclear area (- 180µm 2 ), suggesting that an average of 7 . 5 a-particles (8 MeV helium ions) per cell nucleus are needed to induce a lethal lesion .
1.
Introduction Epidemiological studies have shown that radon and its progeny may play an important role in the lung cancer problem of uranium miners (NCRP 1984) . Recent findings showing high levels of radon in many homes have caused great concern as to the potential risk of radon, and have increased interest in the biological effects of a-particles (Stranden 1980) . Alpha-particles released during the decay of radon progeny are implicated as an important factor in lung cancer induction (NCRP 1984) . Because 85-90°,10 of all cancers are epithelial in origin, a better understanding of the biological effects of a-particles in human lung will result from the study of the radiation response of human airway epithelial cells directly . Recent developments in the culture of human airway epithelial cells have now made such studies possible (Lechner and LaVeck 1985, Gruenert et al. 1988) . We initiated studies on the lethal effects of X-rays and energetic a-particles in human bronchial and tracheal epithelial cells in order to fill an important gap in our knowledge of the radiation response of human airway epithelial cells . This paper reports the results obtained with primary cultures and airway epithelial cells that were transformed with an origin-defective simian virus 40 (SV40) containing plasmid (Gruenert et al . 1988) . 2.
Materials and methods
2 .1 . Cell culture All cultures were human in origin and derived from human trachea or bronchus following autopsy or termination of pregnancy . The isolation and culture of human 0020-7616/90 $3 .00 .~ ; 1990 Taylor & Francis Ltd
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airway epithelial cells has been reported in detail elsewhere (Gruenert et al. 1990) . Briefly, tissue was processed within 24 h of removal . All airway tissue was washed with cold (4°C) phosphate-buffered saline (PBS) containing penicillin (100 U/ml), streptomycin (100µg/ml) and amphotericin B (50µg/ml) . Tracheal and bronchial epithelial cultures were initiated for tissue explants . Explant cultures were established by cutting tissue into 2 mm square pieces and then placing them on tissue culture dishes coated with fibronectin (Collaborative Research Inc .), vitrogen 100 (Collagen Corporation, Palo Alto, CA) and bovine serum albumin (Biofluids, Rockville, MD) . The dishes were coated with a solution containing 1 mg fibronectin, l ml vitrogen 100, 10 ml of 10% bovine serum albumin stock solution and 100 ml basal growth medium (FN-V-BSA) . The coating solution was incubated in the dishes at 37 ° C for 3 h . After incubation the coating solution was removed, and the dishes were dried at room temperature before use . Explants were covered with dialysed serum to facilitate attachment and incubated for 24 h at 37°C under 5 % CO 2 in air . Serum was then diluted with serumfree medium . The serum-free medium is a modified form of LHC-9 medium (Lechner and LaVeck 1985) containing glutamine and no retinoic acid and is designated MLHC-8e (Gruenert et al . 1990) . LHC-8 (Biofluids) is supplemented with epinephrine (0 . 5 µg/ml) . This LHC-8e medium was further modified (Gruenert et al . 1990) . The Stock 4 and trace element components of the medium were increased by 20% . The final concentration of glutamine was 4mM . Since blood-derived serum contains tumour growth factor-/3 (TGF-fl), an agent that induces squamous cell differentiation, and because the growth of contaminating fibroblasts is inhibited in serum-free medium (Lechner and LaVeck 1985), cells were put into serum-free medium as soon as possible after isolation (about 48 h after explanting) . After epithelial outgrowth appeared, explants were transferred to other dishes to generate additional epithelial cultures . Explant cultures are often mixed populations of epithelial cells and fibroblasts . Generation of cultures that are exclusively epithelial was achieved by selective trypsinization (Noyes et al . 1980, Gruenert et al . 1990) . The mixed cultures were washed twice with a HEPES buffered saline (HBS) (18 . 3 mm HEPES, 121 mm NaCl, 2-7 mm KCI, 9-4 mm glucose, 7 . 2 mm Na 2 HPO 4 -7H 2 0), phenol red (0 . 5%), 0 . 25 ml/l ; (pH 7 . 45) (Lechner and LaVeck 1985 ; Gruenert et al . 1990) . The cells were then incubated at 37°C in a trypsinization cocktail containing 0 . 02% EGTA, 1 % polyvinylpyrrolidine (PVP), and 0 . 02% trypsin, 0 .0016% EDTA in Puck's saline A and 80 mg/l glucose . Fibroblasts were detached by rinsing once with the trypsinization solution followed by two additional rinses with HBS . Attached cells were incubated at 37°C overnight in the presence of MLHC-8e medium under 5% CO 2 in air and assessed the following day for further fibroblastic contamination . Selective trypsinization was repeated within 48 h of the initial trypsinization if there was still morphological evidence of fibroblasts . Cells were subcultured using the same trypsinization solution as described above when they reached 80-90% confluence . Cultures were rinsed twice with HBS, and then incubated at 37°C in the trypsinization solution . The cells were then pelleted and resuspended in MLHC-8e medium . New cultures were established at a split ratio of between 1 : 2 and 1 :4 on tissue culture plastic coated with FN-V-BSA as described above . Growth medium was routinely replaced with fresh LHC-8e every other day . Transformed tracheal epithelial cells were obtained by transfecting the primary
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cultures with a plasmid containing an SV40 virus defective in the origin of replication (pSVori-) (Gruenert 1987, Gruenert et al . 1988) . The post-crisis 9HTEo line has grown well in Eagle's MEM supplemented with 10% fetal calf serum, glutamine and antibiotics . The 9HTEo cells are immortal and are no longer contact-inhibited . They also exhibit anchorage-independent growth by forming colonies in soft agar . No tumorigenicity was indicated after injecting 10 6 cells into athymic nude mice (unpublished observations) . The pre-crisis cells used for survival studies were obtained a few passages after pSVori- transfection . These cells were grown and subcultured in the same way as cultures of non-transformed epithelial cells . The epithelial origin of the cells was determined immunocytochemically . All cultures were assayed for the presence of keratin, an epithelial-specific marker (Gruenert et al . 1988, 1990) . The presence of epithelial-specific markers such as keratin, tight junction formation, microvilli containing actin-like filaments, and ion transport has been previously verified for cultures of both transformed and nontransformed epithelial cells used in these studies (Gruenert et al. 1988, 1990, respectively) . 2 .2 . Irradiation The Super HILAC at LBL provided the monoenergetic helium beams for all the radiation studies . Routine beam monitoring and exposure dosimetry were done by using a thin-walled parallel plate ionization chamber immediately downstream of the beam exit window and adjacent to the sample exposure position . The calibration of the ion chamber was made by means of a beam total-charge collector (Faraday cup) located at the sample exposure position . The energy of the helium beam was determined with a silicon detector . The uniformity of the beam within the irradiated sample area and the accuracy of dose was within 5% . About 10-12 h before irradiation, exponentially growing stock cells were dissociated with a trypsinization solution containing PVP, EGTA, trypsin and EDTA (Gruenert et al . 1988) . Cells were plated at a density giving about 50 colonies per dish for each treatment dose . These dishes were checked for multiplicity shortly before radiation exposure . In these studies very few doublets were found, possibly due to the long doubling time (,> 36 h) . Cell growth medium was removed from the dish just before irradiation . In order to minimize the problem of cell drying the dose rate was carefully adjusted so that the irradiation time for all doses used was less than 20 s . Immediately after irradiation, fresh medium was added to the cells, and the dish was then incubated for 2-3 weeks at 37°C to allow colony formation . At the end of the incubation period, cells were fixed and stained, and colonies containing more than 50 cells were counted as survivors . The present study thus used only the standard clonogenic assay . The possible induction by radiation of lethal mutations in the progenies of survivors, as found by other investigators (Seymour et al . 1987, Gorgojo and Little 1989), were not studied . For radiation experiments with primary cultures and pre-crisis cells, fibronectin-coated dishes and feeder cells were used . About 5 x 10 4 mouse fibroblasts, given 50 Gy of X-rays, were seeded into each dish as the feeder cells . Four dishes were used for each radiation dose, and six dishes for the control . X-ray experiments were performed with a 250 kVp Philips X-ray machine, operated at 225 kVp, 15 mA, with 0 . 35 Cu filter and a dose rate of 85 cGy/min . Tissue culture dishes (60 mm) were irradiated on a rotating table, and the uniform-
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ity of dose over the whole area of dish was better than 3%, as measured by a Victoreen ionization chamber . 2 .3 . Data analysis Survival data obtained from these experiments were fitted by a computer program, using the least-squares method, to determine the a and /3 values for the linear-quadratic model and n and D o for the multi-hit and single-target model . The standard errors for the survival data were calculated from the formula SE=(n) t12 /N, where n is the total number of colonies counted and N is the number of plates used . The cross-section was calculated using the following equation : a (µm2)=[0 .16 x LET (keV/um)]/Do (Gy) where a is the cross-section and D o is obtained from the final slope of the survival curve (Curtis 1988) . 3.
Results Figure 1 shows the results for the immortal 9HTEo cells irradiated by X-rays and 8 MeV helium ions . The X-ray survival curve showed a significant shoulder (extrapolation number n of 3 . 2) and a D o about 120cGy . The survival curve for
I
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I
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,tt
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I0 N 0
X RAYS
`
RBE : 2 .4 ~10
5 .tt 2 I
I
0
2
6 4 DOSE (Gy)
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Figure 1 . Dose-response curves for survival of immortal human airway epithelial cells, post-crisis, P3-83 (9HTEo) irradiated by X-rays and 8 MeV helium ions . The average plating efficiency was 30% .
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helium ions appeared to be close to exponential with a D o about 59 cGy . The RBE determined by the ratio of D o for X-rays to that for helium ions was 2-0 (Table 1) . The RBE determined at 10% survival level, however, was slightly higher, about 2-4 . The survival curves for primary cultures are shown in Figure 2 . For X-rays the n and D o were 1-86 and 128 cGy, respectively . The extrapolation number and D o for helium ions were 1-67 and 57-5 cGy, respectively . The RBE determined by the final slope was 2-2, similar to that measured at 10% survival level (2 . 3) . The X-ray survival curve for pre-crisis cells indicates an n of 2 . 35 and a Do of 142 cGy (Figure 3) . The n and Do for helium ions was 1 . 55 and 79 cGy, respectively . The RBE was 1-79 as determined by the final slopes and 2 .1 at 10% survival level . The a-values for all survival curves show a 2-3-fold increase for cells irradiated by helium ions, as compared with those irradiated by X-rays (Table 1) . For primary cultures, for example, the a was 435 x 10 -3 cGy - ' and 11-17x 10 -3 cGy - ' for X-rays and helium ions, respectively . The fl-value of X-rays was very similar for -2 . primary, pre-crisis and post-crisis cells, about 4-6 x 10 -6 cGy The #-value for helium ions showed a large standard deviation, suggesting that the survival curves, especially for primary and post-crisis cells, might be fitted equally well by a pure exponential function . For determining the probability of interaction between the helium ions and the target in the cell we have calculated the cross-sections . The cross-sections for helium ions, calculated from the Do values, indicated no significant difference between primary culture and post-crisis cells, at about 24 µm 2 (Table 1) . The crosssection of pre-crisis cells was about 17-5µm 2 , somewhat smaller than that for primary culture and post-crisis cells . 4.
Discussion Progress in quantitatively understanding the radiation response of human epithelial cells has been limited by the difficulty of growing these cells in vitro . Table 1 . Inactivation of human airway epithelial cells by helium ions and X-rays
Cells Primary
Number Alpha Beta of experi(x 10 - 3 (x 10 -6 2) ments Radiation cGy - ') cGy_ 2
X-rays He
Precrisis
2
X-rays He
Postcrisis
3
X-rays He
n
o (cGy) D
4 . 35 ±0-58 11-17 +2 . 98
4-66 +1 . 61 18-06 +16 . 23
1 . 86 +0 . 38 1-67 +0 . 47
128-5 +3 . 1 57 . 7 +5 . 2
2-96 ±0 . 32 7 . 25 _ 0 . 84 + 2-65 +0-51 12-58 ±2 . 38
4-43 ±0 . 57 16-1 +448 6-51 +1-04 10-34 ±12 . 87
2-35 ±0 . 42 1-55 _ 0 . 22 + 3 . 21 +0-62 1 . 53 ±0 .43
141-8 ±7 . 1 79-4 +4 .8 119-9 +5-7 59-2 ±5 . 5
* Dox is D o for X-rays, and Do„, for helium ions .
RBE (Do , / Do „.)*
Crosssection (µm 2 )
1-0 2-23 +0 . 05
24-2 +2 . 0
1 .0 1-79 +0 . 08 1 .0
17-5 +1 . 0
2 . 03 ±0 . 09
23-5 ±2 . 0
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100 t.
2;{
X RAYS
O
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4
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50
J Q
20 , RBEi o 2 .3
I0 U) 5
2
I 0
2
4 DOSE
6 (Gy)
8
10
Figure 2 . Survival curves for primary culture of human airway epithelial cells irradiated by X-rays and 8 MeV helium particles . The average plating efficiency was 15°c . Recent advances in epithelial culture have facilitated assessment of the radiosensitivity of several types of human epithelial cells . Human mammary (Gathers and Gould 1983, Yang et al. 1983, 1989), thyroid (Miller et al. 1987, Seymour and Mothersill 1987), epidermis (Dover and Potten 1983, Kasid et al . 1987) and umbilical cord vein (Hei et al . 1987, Hall et al . 1988) epithelial cells have all been studied using clonogenic assays . Our results with human airway epithelial cells were very similar to those reported for epidermal keratinocytes (Kasid et al . 1987), i .e . no significant difference in radiosensitivity between primary cultures and established cells . There is, however, a significant difference in D o between airway epithelial cells (D o = 120-130 cGy) and epidermal keratinocytes (D o =224-243 cGy) . This difference is not totally unexpected since different D o values have been observed for thyroid and mammary gland epithelial cells . Whether this difference in radiosensitivity between cells from different tissues is due to differences in repair mechanisms or other epigenetic factors, e .g . extracellular matrix, is unknown at present . Investigators at several laboratories have determined the RBE values for a-particles with various energies in various cell systems . For human fibroblasts, for example, RBE values ranging from 1 . 4 to 4 . 0 have been reported for accelerated helium ions with LET from 20 to 90keV/µm, respectively (Cox et al . 1977) . The lethal effect of a-particles with LET of 110-200keV/µm was studied with human T-1 cells, and an RBE of 8 was found at low doses of a-particles with
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50
10
t3
X RAYS
0
He4
515
-RBE ; 2 .1 10
0 5 tt
2
I
0.5 0
2
4 DOSE
6
8
I0
(Gy)
Figure 3 . Survival curves for pre-crisis human airway epithelial cells irradiated by X-rays and 8 MeV helium ions . The average plating efficiency was 21 % .
an LET of 110 keV/µm (Barendsen 1964) . Our studies with 8 MeV helium ions (- 87keV/µm) showed that the RBE for primary and established human tracheal epithelial cells was 2 . 2-2 . 4 and is in the range of RBE values reported for other human cell systems . Previous studies have suggested that cells in the more advanced stages of neoplastic progression have increased sensitivity to low-LET radiation (Gantt et al . 1987, Sanford et al. 1987) . Our observations of cell survival show no difference in X-ray or a-particle sensitivity between cells in progressive stages of transformation or non-transformed cells . These results are supported by those found by Bender et al . (1988) . Using the experimental data, we calculated the cross-sections for cell inactivation . Use of the D o of the final slope of the survival curve to calculate the crosssection is valid for an exponential curve with no change of slope . The cross-section calculated from the D o of a shouldered survival curve, in general, will be larger than that from the initial slope which gives the true cross-section value . Because the survival curve of helium ions has only a very small shoulder, i .e . is nearly exponential, the calculated cross-section value should not differ significantly from the true value .
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The cross-section for human airway epithelial cells, determined from the helium ion survival curves, was about 24pm 2 , which is significantly smaller than their average geometric nuclear area (-180 µm 2 ) . These results suggest that an average of 7 . 5 helium ions per cell nucleus are needed to induce a lethal lesion . Therefore, some 85% of the helium ions traversing a cell nucleus do not result in lethality . A single traversal of an a-particle in the cell nucleus, therefore, could cause some non-lethal effects, such as mutations and/or chromosomal aberrations . At present it is unknown whether a single a-particle traversing the cell nucleus is sufficient to induce neoplastic transformation . Understanding the carcinogenic
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effect of a-particles in human airway epithelial cells, especially at low doses, is particularly important for assessing the potential cancer risk of radon exposure in the domestic environment . Neoplastic cell transformation experiments with human airway epithelial cells are now under way in our laboratory . Acknowledgements The expert typing and editing of Tennessee Gock is appreciated . We also thank Laurie Craise and Sujay Dutta for their technical assistance . This research is supported by the Office of Health and Environmental Research, US Department of Energy, under Contract DE-AC03-76SF00098, and NIH Grant DK39619 (DCG) . References BARENDSEN, G . W., 1964, Impairment of the proliferative capacity of human cells in culture by alpha particles with differing linear-energy transfer . International Journal of Radiation Biology, 8, 453-466 . BENDER, M . A ., VIOLA, M . V ., FIORE, J ., THOMPSON, M . H . and LEONARD, R . C ., 1988, Normal G 2 chromosomal radiosensitivity and cell survival in cancer familial syndrome . Cancer Research, 48, 2579-2584 . CATHERS, L . E . and GOULD, M . N ., 1983, Human mammary cell survival following ionizing radiation . International Journal of Radiation Biology, 44, 1-16 . Cox, R ., THACKER, J ., GOODHEAD, D . T . and MUNSON, R . J ., 1977, Mutation and inactivation of mammalian cells by various ionizing radiations . Nature, 267, 425-427 . CURTIS, S ., 1988, Radiation physics related to biology . Terrestrial Space Radiation and Its Biological Effects, edited by P . D . McCormack, C . E . Swenberg and H . Bucker (Plenum, New York), pp . 301-313 . DOVER, R . and POTTEN, C . S ., 1983, Radiosensitivity of normal human epidermal cells in culture . International Journal of Radiation Biology, 6, 681-685 . GANTT, R ., SANFORD, K . K ., PARSHAD, R ., PRICE, F . M ., PETERSON, W . D ., JR and RIHM, J . S ., 1987, Enhanced G 2 chromatic radiosensitivity at an early stage in the neoplastic transformation of human epidermal keratinocytes in culture . Cancer Research, 47, 1390-1397 . GoRGOJo, L . and LITTLE, J . B ., 1989, Expression of lethal mutations in progeny of irradiated mammalian cells . International Journal of Radiation Biology, 55, 619-630 . GRUENERT, D . C ., 1987, Differentiated properties of human epithelial cells transformed in vitro . Biotechniques, 5, 740-748 . GRUENERT, D . C ., BASBAUM, C . B ., WELSH, M . K ., LI, M ., FINKBEINER, W . E . and NADEL, J . A ., 1988, Characterization of human tracheal epithelial cells transformed by an origin-defective SV40 . Proceedings of National Academy of Sciences, USA, 85, 5951-5955 . GRUENERT, D . C ., BASBAUM, C . B . and WIDDICOMBE, J . M ., 1990, Long term culture of normal and cystic fibrosis epithelial cells grown in serum free medium . In Vitro, 96, 411-418 . HALL, E . J ., MARCHESE, M ., HEi, T . K . and ZAIDER, M ., 1988, Radiation response characteristics of human cells in vitro . Radiation Research, 114, 415-424 .
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HEI, T . K ., MARCHESE, M . J . and HALL, E . J ., 1987, Radiosensitivity and sublethal damage repair in human umbilical cord vein endothelial cells . International Journal of Radiation Oncology, Biology, Physics, 13, 879-884.
KASID, U . N ., RHIM, J . and DRITSCHILO, A ., 1987, Human epidermal keratinocytes retain radiation resistance following in vitro immortalization and malignant transformation . Radiation Research, 111, 565-571 .
LECHNER, J . F . and LAVECK, M . A ., 1985, A serum-free method for culturing normal human bronchial epithelial cells at clonal density . Journal of Tissue Culture Methods, 9,
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43-48 .
MILLER, R . C ., HIRAOKA, T ., KOPECKY, K . J ., NAKAMURA, N ., JONES, M . P ., ITO, T . and CLIFTON, K . H ., 1987, Sensitivity to radiation of human normal, hyperthyroid, and neoplastic thyroid epithelial cells in primary culture . Radiation Research, 111, 81-91 . NCRP, 1984, Evaluation of Occupation and Environmental Exposures to Radon and Radon Daughters in the United States . NCRP report No . 78 (National Council on Radiation Protection and Measurement) . NOYES, I ., MILL, G . and CUNNINGHAM, C ., 1980, Establishment of proliferating human epithelial cells in vitro from cell suspension of neonatal foreskin . Tissue Culture Association Manual,
5, 1173-1175 .
SANFORD, K . K ., PARSHAD, R ., GANTT, R ., TARON, T . E ., JOHNS, G . M . and PRICE, F . M ., 1987, Factors affecting and significance of G Z chromatin radiosensitivity in predisposition in cancer . International Journal of Radiation Biology, 51, 381-391 . SEYMOUR, C . B . and MOTHERSILL, C ., 1987, Long-term effects of gamma irradiation on cultured human thyroid cells . International Journal of Radiation Biology, 51, 381-391 . SEYMOUR, C . B ., MOTHERSILL, C . and ALPER, T., 1987, High yields of lethal mutations in somatic mammalian cells that survive ionizing radiation . International Journal of Radiation Biology, 50, 167-179 .
STRANDEN, E ., 1980, Radon in dwellings and lung cancer-a discussion . Health Physics, 38, 301-306 .
YANG, T . C ., STAMPFER, M . R . and SMITH, H . S ., 1983, Response of cultured normal human mammary epithelial cells to X-rays . Radiation Research, 96, 476-485 . YANG, T . C ., STAMPFER, M . R . and TOBIAS, C . A ., 1989, Radiation studies on sensitivity and repair of human mammary epithelial cells . International Journal of Radiation Biology, 56,605-609 .
