Oncogenic Transformation of Mammalian Cells in vitro with Split Doses of X-Rays

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  • Oncogenic Transformation of Mammalian Cells in vitro with Split Doses of X-RaysAuthor(s): Richard C. Miller, Eric J. Hall and Harald H. RossiSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 76, No. 11 (Nov., 1979), pp. 5755-5758Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/70537 .Accessed: 08/05/2014 04:26

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  • Proc. Natl. Acad. Sci. USA Vol. 76, No. 11, pp. 5755-5758, November 1979 Cell Biology

    Oncogenic transformation of mammalian cells in vitro with split doses of x-rays

    (dose-response relationship/low-dose irradiation/estimation of cancer risk)


    Radiological Research Laboratory, Department of Radiology, and the Cancer Center/Institute of Cancer Research, Columbia University College of Physicians and Surgeons, New York, New York 10032

    Communicated by Michael Kasha, August 31, 1979

    ABSTRACT An established line of mouse fibroblasts, C3H/1OT1/2 cells, was used for the assessment in vitro of onco- genic transformations caused by single and split doses of x-rays. The shape of the dose-response relationship was determined over the range from 0.1 to 10 Gy. It was found that splitting the x-ray dose into two equal fractions, separated by 5 hr, led to a reduction in transformation frequency at doses above 1.5-2 Gy but to an enhancement of transformation at lower doses. The observations reported cast doubt on the assessment of human cancer risk at low dose levels by a linear extrapolation from available high-dose data from the Japanese atomic bomb sur- vivors or from persons exposed for medical purposes.

    In a previous paper (1), we reported preliminary data showing that, at low dose levels, splitting an x-ray dose into two equal fractions enhanced transformation frequency compared with the same total dose delivered in a single exposure, whereas at higher dose levels fractionation produced the more conventional sparing effect. The effect on transformation of fractionating low x-ray doses is of such fundamental and practical importance that we considered it imperative to accumulate much more data, to subject them to a rigorous statistical analysis in order to allow unequivocal conclusions to be drawn, and to extend the scope of the experiments to even lower dose levels in order to elucidate the shape of the dose-response relationship.

    The importance of the observations reported here lies in their possible implication to the development of human cancer risk estimates at low doses, by extrapolation from available data relating to high dose levels. Both major reports to appear in recent years, the UNSCEAR report of the United Nations (2) and the BEIR report of the U.S. National Academy of Sciences (3), use a linear extrapolation. The risk estimates assumed to apply at low doses are calculated from the slope of a straight line drawn from the origin through the data points for excess cancer incidence for higher doses, usually in excess of 100 rem. Furthermore, it is assumed in both reports that the linear ex- trapolation leads to an upper limit for the risk estimation at low doses that is "conservative" and "prudent," because most high-dose data in the human relate to single acute exposures, while the low-dose exposure of the public from man-made ra- diations is the result of multiple small exposures, which are assumed to be less effective.


    The C3H/10T'/2 mouse fibroblast cell line was used for these experiments. Isolated in the laboratory of Charles Heidelberger, these cells exhibit good contact inhibition after confluence, unless treated with chemicals or radiation, in which case a small proportion of the cells grow into dense piled-up clones that are

    The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- vertisement " in accordance with 18 U. S. C. ?1734 solely to indicate this fact.



    FIG. 1. Type III clone of transformed cells showing the dense piled-up cells and the criss-cross pattern at the edges. The confluent layer of contact-inhibited untransformed cells can be seen in the background.

    capable of producing tumors when injected into compatible animals (4, 5). The details of the procedures have been pub- lished (6). Briefly, cells were seeded at low density into 50-cm2 petri dishes such that an estimated 400 reproductively viable cells would survive the subsequient irradiation. Cells were al- lowed to attach overnight at 37?C for about 18 hr before being exposed to x-rays. The cells were at room temperature during irradiation, but were returned to a 37?C incubator between split doses. After all x-ray treatments the cells were incubated for 6 weeks, with the growth medium changed twice weekly, to allow the transformations to be expressed and to grow into visible clones. At the end of this period the cells were fixed with formalin and stained with Giemsa stain; type II and type III foci were scored as transformed, using the criteria described by Reznikoff et al. (5).


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  • 5756 Cell Biology: Miller et al. Proc. Natl. Acad. Sci. USA 76 (1979)

    4 I I



    0 2 4 ~ ~ ~~6 8 Time between doses, hr

    FIG. 2. Influence on transformation rate of the time interval between two dose fractions of 0.5 Gy of x-rays: The results from two experiments are shown, sSD.

    Fig. 1 shows a typical type III clone that can be readily identified by the densely stained piled-up appearance of the cells and the criss-cross pattern at the periphery of the clone, which shows up clearly against the background of lightly stained contact-inhibited untransformed cells.

    Irradiations were performed with a Siemens Stabilipan x-ray therapy unit, operated at 300 kV (peak), 12 mA, with added filtration of 0.2 mm Cu. For the higher x-ray doses, a treatment distance of 50 cm was used, at which the dose rate was com- puted to be 1.8 Gy/min. For the lower x-ray doses, a longer treatment distance of 118 cm was used, at which the dose rate was 0.32 Gy/min. In all cases the exposure time was less than 5 min. The longer treatment distance was used at lower doses to allow a larger number of dishes to be irradiated simulta- neously; this was necessary because, as can be seen from Table

    105: | | 'it III I 111111 li




    0.1 ~~~1 10 Dose, Gy

    FIG. 3. Pooled data from many experiments for the transfor- mation rate for single (0) and split (i) doses of x-rays. The time in- terval between split doses was 5 hr.

    1, a typical experiment at low dose levels involved over 1000 petri dishes.


    Transformation frequencies produced by two doses of x-rays of 0.5 Gy, separated by a time interval from 0 to 7.5 hr, are shown in Fig. 2. Fractionation leads to an elevated incidence of transformation, which increases with increasing time interval between the doses up to a maximum at about 4-5 hr.

    The accumulated data from experiments designed to com- pare the transformation frequencies after single and split x-ray doses are summarized in Table 1 and plotted in Fig. 3. The design of the experiments was such that single and split doses were always compared within a given experiment. Because of the sheer size of the experiments, and the number of dishes involved, only a limited number of doses could be used in a given experiment. This was particularly true at the lower end of the dose scale. An interesting pattern emerges. At doses above about 2.0 Gy, fractionation leads to a reduction in transfor- mation frequency compared with a single exposure of the same total dose. Between 0.3 and 1.5 Gy, fractionation enhances the incidence of transformation.

    The cell survival data for single and split doses, resulting from the same experiments in which transformation was scored, are shown in Fig. 4. In all cases, fractionation leads to a sparing as far as cell lethality is concerned.


    Two interesting and potentially important results emerge from the present investigation, one a direct consequence of the other: (i), the shape of the dose-response relationship for transfor- mation; (ii) the effect of fractionation on the frequency of transformation, which varies with dose. These need to be dis- cussed in turn.

    First, the shape of the dose-response relationship. In Fig. 3,


    c \ \ Split o dose

    Single dose


    0.1 \

    0 2 4 6 8 10 Dose, Gy

    FIG. 4. Survival data for cells exposed to single and split doses of x-rays. The time interval between split doses was 5 hr.

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  • Cell Biology: Miller et al. Proc. Natl. Acad. Sci. USA 76 (1979) 5757

    Table 1. Frequency of transformation induced by various single and split doses of x-rays

    Single dose Split dose (5 hr) Average Average

    transformation transformation Dose, Surviving Transformation frequency Surviving Transformation frequency

    Exp. Gy Dishes fraction frequency* X 104t Dishes fraction frequency* X 104t

    1 0.10 119 1.00 4/ 63,300 100 0.97 4/ 57,000 2 114 0.95 8/ 88,800 0.686 ? 0.135 110 0.99 7/ 89,300 0.637 ? 0.133 3 350 9.98 14/227,000 338 0.99 12/214,600

    4 0.30 62 0.91 5/ 40,400 94 0.91 15/ 60,000 5 167 0.91 15/101,000 1.40 ? 0.23 166 0.94 31/109,000 2.83 + 0.33 6 215 0.88 18/131,000 185 0.91 29/ 96,200

    7 0.50 59 0.91 4/ 29,200 93 0.90 14/ 49,300 8 78 0.98 5/ 38,600 92 0.98 12/ 57,900 9 191 0.87 14/ 96,500 1.52 ? 0.18 128 0.98 21/ 72,700 2.78 : 0.28

    10 198 0.89 25/155,000 135 0.97 36/115,000 11 132 0.98 11/ 60,200 150 0.98 18/ 68,400

    6 1.00 150 0.92 10/ 61,700 1.76 + 0.41 114 0.97 13/ 48,300 2.86 +0.62 11 100 0.93 8/ 40,500 69 0.91 8/ 25,100

    5 2.00 51 0.69 7/ 18,400 50 0.87 4/ 10,800 12 33 0.66 3/ 11,400 3.27 + 0.73 85 0.67 2/ 9,600 2.94 + 0.74 14 87 0.59 10/ 31,100 102 0.62 10/ 34,000

    7 3.00 68 0.47 6/ 9,800 126 0.76 14/ 52,000 8 131 0.39 28/ 55,000 4.55 ? 0.63 133 0.72 23/ 53,800 3.76 ? 0.52

    10 130 0.50 18/ 49,500 92 0.71 16/ 35,200

    9 4.00 23 0.31 8/ 5,200 17 0.40 3/ 2,400 12 81 0.30 34/ 32,800 996 i 1.17

    61 0.39 16/ 22,000 7.04 + 1.07 13 43 0.43 13/ 14,700 9 * 30 0.49 9/ 13,300 14 52 0.26 17/ 19,600 52 0.48 15/ 23,400

    12 8.00 17 0.051 9/ 2,500 21 0.13 3/ 3,700 13 31 0.053 30/ 9,600 24.4 ? 2.7 36 0.16 17/ 10,900 12.0 i 1.90 14 63 0.060 44/ 21,900 68 0.19 22/ 20,400

    12 10.00 27 0.021 31/ 9,700 31.9 + 5.7 43 0.10 20/ 15,000 13.3 ? 3.00

    * Number of transformed clones/total surviving cells. t Averages + 1 SD are given.

    transformation frequency is plotted against dose on a double logarithmic scale. The slope clearly changes over the range of doses used. At higher doses, above about 2 Gy, the curve is steep and certainly consistent with a slope of 2, implying that trans- formation frequency may be related to the square of the ab- sorbed dose. At lower doses, below about 0.3 Gy, the curve has a slope consistent with unity, implying that the transformation frequency may be directly proportional to dose. Over the in- termediate dose range, the curve is shallow indeed, and within the confidence limits of the data points, transformation fre- quency barely changes at all between 0.3 and 1 Gy.

    Second, a complex pattern emerges for the effect of frac- tionation on transformation. Above about 1.5-2 Gy, dividing a given dose into two equal fractions results in a reduction in transformation. Between 0.3 and 2 Gy, fractionation clearly results in an elevated frequency of transformation. This results directly from the changing slope of the dose-response rela- tionship for single exposures. Indeed, the dose-response curve for split doses can be derived from that for single exposures if it is assumed that the two exposures, 5 hr apart, are totally in- dependent and do not interact with one another in any way. On this basis, the transformation frequency for two doses of D/2 Gy, separated by 5 hr, should be twice that for a single exposure

    of D Gy. This is found to be approximately true over the entire range of doses tested. The maximum separation between the curves is a factor of 2 in transformation frequency between single and split exposures of the same total dose. The data re- ported here all involve C3H/1OT'/2 cells, but the effects of fractionation have been reported by Borek and Hall (7) and by Borek (8) for cells derived from fresh explants of hamster em- bryos. The data are given in Fig. 5, in which, to facilitate comparison, the ratio of transformation frequencies for split to single doses is plotted as a function of dose. Compared in this way, there is a remarkable similarity in the effects of frac- tionation on transformation assessed by these different bio- logical systems. Above 1.5-2 Gy, fractionation decreases transformation frequency, whereas below this dose level frac- tionation enhances it.

    It is evident, then, that in the case of in vitro transformation, the dose-response relationship has a sufficiently complex shape, so that the transformation frequency for low doses cannot be predicted accurately by a linear extrapolation from data ob- tained at high doses. It can be seen from Fig. 3 that a linear extrapolation from high doses may either substantially over- estimate, or equally well underestimate, the transformation incidence at low doses, depending upon the dose range in-

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  • 5758 Cell Biology: Miller et al. Proc. Nati. Acad. Sci. USA 76(1979)



    ,) /


    4., ~ ~ ~ ~ Doe IGy

    4- F

    0.1 1

    Dose, Gy

    FIG. 5. Comparison of the data presented in this paper for C3H/1OT1/2 cells (o) with the data of Borek and Hall (7) and Borek (8) for fresh explants of hamster embryo cells (0). The ratio of the transformation frequencies for split and single doses is plotted as a function of total dose. A ratio in excess of unity implies that frac- tionation enhances transformation; a ratio of less than unity implies that fractionation results in a reduction of the transformation rate. For both cell systems the crossover point between the enhancing and sparing effect of fractionation occurs at about 1.5-2.0 Gy.

    volved. Furthermore, there is a broad and important range of doses over which fractionation enhances transformation fre- quency so that estimates from data relating to a single prompt exposure do not necessarily represent an upper limit to the transformations that could accrue from multiple small doses.

    It must be admitted, of course, that morphologically iden-

    tified transformed clones in a petri dish are a far cry from leukemia or solid tumors in humans; as a model system, trans- formation in vitro clearly has its limitations. Cell transformation is likely to be an initial step in carcinogenesis, but the transition from transformation of a cell to the development of a tumor is undoubtedly a complex process. It could be argued that dose and dose-rate characteristics of the basic transformation process may be obscured, or even reversed, in the final expression of carcinogenesis.

    However, the attraction of the in vitro transformation system lies in its exquisite sensitivity, as a result of which it is possible to obtain a dose-response relationship over a range of doses, and with a precision, that is unlikely ever to be equaled in humans. It is clearly not prudent to ignore the possible implications of the shape of this dose-response relationship or the enhancement of transformation resulting from fractionation at low dose levels.

    This investigation was supported by Grant CA 23952 and CA 12536 awarded by the National Cancer Institute, and by Contract EP-78- S-02-4733 from the Department of Energy.

    1. Miller, R. & Hall, E. J. (1978) Nature (London) 272,58-60. 2. United Nations Scientific Committee on The Effects of Atomic

    Radiation [UNSCEAR] (1977) Report to the General Assembly on Sources and Effects of Ionizing Radiations (United Nations, New York).

    3. Advisory Committee on the Biological Effects of Ionizing Radia- tions [BEIR] (1972) The Effects on Populations of Exposure to Low Levels of Ionizing Radiation (Natl. Res. Coun., Natl. Acad. Sci., Washington, DC)

    4. Reznikoff, C., Brankow, D. W. & Heidelberger, C. (1973) Cancer Res. 33, 3231-3238.

    5. Reznikoff, C. A., Brankow, D. W. & Heidelberger, C. (1973) Cancer Res. 33, 3239-3249.

    6. Miller, R. C. & Hall, E. J. (1978) Br. J. Cancer 38, 411-417. 7. Borek, C. & Hall, E. J. (1974) Nature (London) 252, 499-501. 8. Borek, C. (1979) Br. J. Radiol. 52, 845-849.

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    Article Contentsp. 5755p. 5756p. 5757p. 5758

    Issue Table of ContentsProceedings of the National Academy of Sciences of the United States of America, Vol. 76, No. 11 (Nov., 1979), pp. 5413-6022Growth of Complex Systems can be Related to the Properties of their Underlying Determinants [pp. 5413-5417]Sulfur Base Ligation to Iron (II) and Cobalt (II) Porphyrins [pp. 5418-5420]Significant Structure Theory Applied to Electrolyte Solution [pp. 5421-5423]Lower Bounds for Eigenvalues of Self-Adjoint Problems [pp. 5424-5425]Phosphorus in Connecticut Lakes Predicted by Land Use [pp. 5426-5429]RNA Ligase Reaction Products in Plasmolyzed Escherichia coli Cells Infected by T4 Bacteriophage [pp. 5430-5434]Molecular Cloning of Human $\epsilon $-Globin Gene [pp. 5435-5439]Implantation of the Isolated Human Erythrocyte Anion Channel into Plasma Membranes of Friend Erythroleukemic Cells by Use of Sendai Virus Envelopes [pp. 5440-5444]Rapid ATP Assays in Perfused Mouse Liver by $^{31}$P NMR [pp. 5445-5449]Binding of Inhibitor Alters Kinetic and 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Murine Cells [pp. 5820-5824]Antibodies to Epstein-Barr Virus-Determined Antigens in Normal Subjects and in Patients with Seropositive Rheumatoid Arthritis [pp. 5825-5828]Human Lymphocyte Antigens: Production of a Monoclonal Antibody that Defines Functional Thymus-Derived Lymphocyte Subsets [pp. 5829-5833]Dissociation and Exchange of the $\beta _{2}$-microglobulin Subunit of HLA-A and HLA-B Antigens [pp. 5834-5838]Amino Acid Sequence of an Immunoglobulin-Like HLA Antigen Heavy Chain Domain [pp. 5839-5842]Disassembly of Viral Membranes by Complement Independent of Channel Formation [pp. 5843-5847]Effect of Interchain Disulfide Bond on Hapten Binding Properties of Light Chain Dimer of Protein 315 [pp. 5848-5852]Structural Characterization of the Murine Fourth Component of Complement and Sex-Limited Protein and their Precursors: Evidence for Two Loci in the S Region of the H-2 Complex [pp. 5853-5857]Induction of Calcium Flux across the Rat Mast Cell Membrane by Bridging IgE Receptors [pp. 5858-5862]Crossreactive Mixed Lymphocyte Reaction Determinants Recognized by Cloned Alloreactive T Cells [pp. 5863-5866]Regulation of the Amplification C3 Convertase of Human Complement by an Inhibitory Protein Isolated from Human Erythrocyte Membrane [pp. 5867-5871]Evidence for a Two-Domain Structure of the Terminal Membrane C5b-9 Complex of Human Complement [pp. 5872-5876]Primary Defect of Insulin Receptors in Skin Fibroblasts Cultured from an Infant with Leprechaunism and Insulin Resistance [pp. 5877-5881]A Practicable Immunological Approach to Block Spermatogenesis without Loss of Androgens [pp. 5882-5885]Neoplastic Transformation of Epithelial Cells in Whole Mammary Gland in vitro [pp. 5886-5890]Retinoid Prevents Mammary Gland Transformation by Carcinogenic Hydrocarbon in Whole-Organ Culture [pp. 5891-5895]Enhancement of Hexose Uptake in Human Polymorphonuclear Leukocytes by Activated Complement Component C5a [pp. 5896-5900]Effect of Glucose/Sulfonylurea Interaction on Release of Insulin, Glucagon, and Somatostatin from Isolated Perfused Rat Pancreas [pp. 5901-5904]Biochemical and Morphologic Studies on Diabetic Rats: Effects of Sucrose-Enriched Diet in Rats with Pancreatic Islet Transplants [pp. 5905-5909]Spontaneous Tumors in Sprague-Dawley and Long-Evans Rats and in their F$_{1}$ Hybrids: Carcinogenic Effect of Total-Body x-Irradiation [pp. 5910-5913]Initiation of Plasma Prorenin Activation by Hageman Factor-Dependent Conversion of Plasma Prekallikrein to Kallikrein [pp. 5914-5918]Triene Prostaglandins: Prostaglandin D$_{3}$ and Icosapentaenoic Acid as Potential Antithrombotic Substances [pp. 5919-5923]Role of Thymidylate Synthetase Activity in Development of Methotrexate Cytotoxicity [pp. 5924-5928]Pyrolysis Products from Amino Acids and Protein: Highest Mutagenicity Requires Cytochrome P$_{1}$-450 [pp. 5929-5933]Changes in Plasma Lipoprotein Distribution and Formation of Two Unusual Particles after Heparin-Induced Lipolysis in Hypertriglyceridemic Subjects [pp. 5934-5938]Galactosamine-Induced Sensitization to the Lethal Effects of Endotoxin [pp. 5939-5943]Antigen-Induced Strain-Specific Autoantiidiotypic Antibodies Modulate the Immune Response to Dextran B 512 [pp. 5944-5947]Reactivation of Herpes Simplex Virus Type 2 from a Quiescent State by Human Cytomegalovirus [pp. 5948-5951]Social Gliding is Correlated with the Presence of Pili in Myxococcus xanthus [pp. 5952-5956]Apolipoprotein is Responsible for Neutralization of Xenotropic Type C Virus by Mouse Serum [pp. 5957-5961]Persistence of Circadian Rhythmicity in a Mammalian Hypothalamic ``Island'' Containing the Suprachiasmatic Nucleus [pp. 5962-5966]Specific Association of Neurotransmitter with Somatic Lysosomes in an Identified Serotonergic Neuron of Aplysia californica [pp. 5967-5971]Distribution of Active and Inactive Forms of Endorphins in Rat Pituitary and Brain [pp. 5972-5976]Widespread Distribution of Protein I in the Central and Peripheral Nervous Systems [pp. 5977-5981]Immunocytochemical Localization, in Synapses, of Protein I, an Endogenous Substrate for Protein Kinases in Mammalian Brain [pp. 5982-5986]Chronic Treatment with Lithium or Desipramine Alters Discharge Frequency and Norepinephrine Responsiveness of Cerebellar Purkinje Cells [pp. 5987-5991]Migration of Schwann Cells and Wrapping of Neurites in vitro: A Function of Protease Activity (Plasmin) in the Growth Medium [pp. 5992-5996]Blockage of Narcotic-Induced Dopamine Receptor Supersensitivity by Cyclo(Leu-Gly) [pp. 5997-5998]Localization of Neurophysin within Organelles Associated with Protein Synthesis and Packaging in the Hypothalamo-neurohypophysial System: An Immunocytochemical Study [pp. 5999-6003]Higher Molecular Weight Forms of Immunoreactive Somatostatin in Mouse Hypothalamic Extracts: Evidence of Processing in vitro [pp. 6004-6008]Intracellular Dye-Marked Enkephalin Neurons in the Magnocellular Preoptic Nucleus of the Goldfish Hypothalamus [pp. 6009-6011]Interrelationships between Ganglionic Acetylcholinesterase and Nonspecific Cholinesterase of the Cat and Rat [pp. 6012-6016]Membrane Potential Changes Caused by Thyrotropin-Releasing Hormone in the Clonal GH$_{3}$ Cell and their Relationship to Secretion of Pituitary Hormone [pp. 6017-6020]Correction: Amino Acid Sequence of Tyrosinase from Neurospora crassa [p. 6021]Correction: Methylation of Herpesvirus saimiri DNA in Lymphoid Tumor Cell Lines [p. 6021]Back Matter


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