238Pu {alpha}-particle-induced C3H10T1/2 transformants are less tumorigenic than the X-ray-induced equivalent

Margaret M. Lehane1,4, Peter E. Bryant2, Andrew C. Riches2, Louise A. Allen1, Cecilie V. Briscoe2, Jean Melville2 and Andrew J. Mill1,3

1 Radiobiology Laboratory, Nuclear Electric Ltd, Berkeley Centre, Berkeley GL13 9PB,
2 School of Biomedical Sciences, University of St Andrews, Bute Medical Building, St Andrews KY16 9TS and
3 Faculty of Applied Sciences, University of the West of England, Frenchay Campus, Coldharbour Lane, Bristol BS16 9TS, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Transformation is a complex multistage process in vitro by which benign cells gradually acquire characteristics of tumour cells. Transformed C3H10T1/2 cells appear in vitro as multilayers of cells termed foci. A variety of transformed phenotypes are observed in vitro and in this study samples of these phenotypes were developed as cell lines and assessed for their ability to induce tumours in C3H mice. It was found that, while a high proportion of X-ray-induced transformants were tumorigenic, most of the {alpha}-particle-induced transformants were non-tumorigenic. Although tumours produced by the X-ray-induced transformants appeared earlier, they grew at similar rates to the {alpha}-particle-induced equivalent. Foci were classified as fully or partially tumorigenic depending on whether the foci produced at least one tumour in the mice injected (partially tumorigenic) or produced tumours in all mice injected (fully tumorigenic). It was found that tumours from the partially tumorigenic foci grew slower or appeared later than those of the fully tumorigenic foci. It is hypothesized that the apparent low tumorigenicity of positively transformed {alpha}-particle-induced foci is due to an increase in genomic instability of progeny focus cells compared with X-ray-induced foci leading to a larger non-viable population of cells in the {alpha}-particle-induced foci.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The C3H10T1/2 mouse cell transformation assay is extensively used as a system to assess the carcinogenic properties of a variety of agents, including chemicals and radiation. Cell transformation is the closest in vitro assay to carcinogenesis in vivo and both are regarded as multistage processes. Transformation in vitro, however, is studied in cells which have already undergone some of the possible steps, for example immortalization, and may be described as the transition of a preneoplastic cell to the neoplastic state. The term transformation is applied to in vitro studies and is distinct from the term carcinogenesis, which applies in vivo, although they may have some common features.

The transformation of a cell involves change(s) which manifests in numerous ways in vitro. Some of these changes include those allowing the growth of cells as tumours in nude mice, those leading to focus formation in vitro and growth of cells in soft agar. The loss of contact inhibition of C3H10T1/2 cells when they become transformed makes the system particularly attractive as a transformation assay. This loss of contact inhibition is observed as a focus of cells which grow in layers. Although the foci are easily distinguished against a monolayer of contact-inhibited cells, the decision as to what constitutes a positively transformed focus is not as easily resolved. The foci produced in the transformation assay were originally classified into three types, designated types I, II and III (1,2). In this original classification type I foci, composed of tightly packed cells, are not scored as positively transformed, since these foci failed to produce tumours in C3H mice. Type II foci show considerable piling up of cells into virtually opaque multilayers with criss-crossing of cells not pronounced. The third category of focus, type III, consists of multilayered criss-crossing arrays of densely staining fibroblastic cells. Type II and III are classified as positively transformed, since it was reported that 50% of type II and 85% of type III produced tumours in C3H mice (1,2). However, not all foci in the C3H10T1/2 assay fall easily into one of these categories and the criteria have been reviewed in attempts to standardize focus categorization (3). A wide range of foci are usually observed with some or all of the above characteristics and classifying the foci as I, II or III often disguises borderline cases. These borderline foci can create significant differences in the transformation data obtained in different laboratories, depending on the manner of scoring of the foci and the criteria deemed most important for the distinction between positively and non-positively transformed foci. This can be a particular problem when quantitative information is compared between different laboratories who operate different criteria.

The most definitive test of positively transformed cells in vitro is the ability of the cells to produce tumours in vivo. However, it is not feasible to isolate and test all the transformed foci produced in a typical C3H10T1/2 assay. Several authors have isolated sample foci and tested the tumorigenicity (Table IGo). The foci from which the cell lines were derived and tested in all these publications were classified into type I, II or III foci, using the criteria of the original authors (1,2). Most of the studies have used chemicals to induce the foci, with >100 foci induced by chemicals tested for tumorigenicity, while data presented for X-ray-induced foci are more limited (48) and even fewer reports are found for {alpha}-particle-induced foci (10). The data presented in these publications illustrate the scale of differences obtained in many laboratories where type II and III foci have been examined for tumorigenicity. These findings also differ from those first reported in the original C3H10T1/2 publication where the tumorigenicity for type II and type III foci was reported as 50 and 83%, respectively (1,2). However, if the data from the studies in Table IGo on foci examined for tumorigenicity in C3H mice (12 studies) are combined, then 45% (10 of 22 foci) of type II foci and 83% (52 of 63 foci) of type III foci were tumorigenic, closely correlating with the original findings (1,2). One of the many variables in the C3H10T1/2 transformation assay is the subjective nature of what are considered to be the important criteria for positively transformed foci. This is especially so since no two foci appear the same and there are a wide range of foci which are borderline between the types I, II and III classifications. Since the calculation of transformation frequencies induced in this system by a variety of carcinogens relies on the identification of transformed foci, it is important to verify the relationship of transformed foci identified in vitro to tumorigenicity in vivo.


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Table I. Published tumorigenicity data of carcinogen-induced C3H10T1/2 focus cell lines (fraction and percentage tumorigenic foci)
 
Furthermore, Table IGo also shows that the number of foci examined is small, less than five, in most cases and even in a large study which examined 75 foci (18) no type I foci were included in the study. Data are presented here on the tumorigenicity of 27 X-ray-induced foci and 58 {alpha}-particle-induced foci. In this study a range of morphologically distinct foci were included and types I, II and III foci were examined.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Transformation and focus isolation
C3H10T1/2 cells, once irradiated, were seeded into culture flasks containing prewarmed and pregassed medium (BME medium, 4 mM L-glutamine, 10% fetal calf serum, 25 ng/ml gentamicin) at cell numbers aimed to provide 2 viable cells/cm2. Parallel flasks to determine survival were also set up. All cultures were placed at 37°C in an atmosphere of 5% CO2 in air. Cells were stained after 10–14 days, depending on growth, and plating efficiency and surviving fractions determined. In the transformation assay the medium was changed first at 2 weeks and then at weekly intervals. The first medium change was with medium supplemented with 10% fetal calf serum; all subsequent changes were with medium supplemented with 5% fetal calf serum. The standard transformation assay continued for 6 weeks after seeding cells. Flasks were then stained with 10% Giemsa stain and examined. In the studies to isolate foci, a standard transformation assay (1,2) was set up with the cultures incubated for 10 weeks rather than the standard 6–7 weeks adopted by many laboratories (for more details see ref. 19). The longer incubation period allowed the foci to grow to a size which made their isolation feasible. Foci to be isolated were photographed the day before isolation while still in culture. The focus isolation procedure first involved the removal of the growth medium from the monolayer followed by a brief wash with fresh growth medium to remove loosely attached cells from the monolayer. A small volume of medium was then added to the culture (~2 ml) and focus isolation accomplished by scraping ~50% of the focus, using a cell scraper, into the medium, which was then aspirated into a separate tissue culture flask (containing growth medium). Most foci remained as clumps of cells which adhered to the cell scraper sufficiently for a gentle transfer of the focus into the medium with minimal disruption of the remainder of the cell monolayer. The focus cells were left to attach and grow and subsequently subcultured into larger tissue culture flasks for further growth to cell numbers which allowed frozen cell stocks of each focus to be established. The remaining culture, from which the focus was originally isolated, was stained with 10% Giemsa stain (BDH) and the focus classified as type II/III or type I, using the stained remainder of the foci and the photographs taken before the foci were isolated. At least eight people examined each focus.

The {alpha}-particle-induced foci (58 foci in total) were isolated in two batches, with a week between batches, while the X-ray-induced foci (27 foci) were isolated together from a single experiment. Foci are classified using the criteria of the original authors (1,2), although no distinction was made between type II and type III foci and the most important criterion for a positively transformed focus was the presence of criss-crossing cells. Since type II and type III foci are regarded as positively transformed when used to assess transformation frequencies in a standard transformation assay (2), it was decided to combine them into one category for the purposes of this study.

Radiation details
For the X-ray exposures a standard constant potential 420 kVp X-ray unit operated at a voltage of 250 kV and a current of 15 mA was used. Parent C3H10T1/2 cells were exposed to a filtered beam (filter containing 1.2 mm aluminium and 0.3 mm copper) while in 25 cm2 culture flasks placed in a perspex phantom. The {alpha}-particle source is located at the Medical Research Council Radiation and Genome Stability Unit (Didcot, UK). Details of the source have been published (20,21). The incident {alpha}-particle energy was 3.26 ± 0.22 MeV and the incident linear energy transfer (LET) in water was 121 keV/µm. The {alpha}-particles traverse a path of 65 mm of helium at atmospheric pressure, a containment chamber window of 0.35 mg/cm2 Hostaphan (polyethylene terephthalate) and 3 mm of air before entering the base (0.35 mg/cm2 Hostaphan) of the culture dish on which the cell monolayer was growing. Up to 10 dishes were mounted in a horizontal wheel, which was rotated continuously at 3 r.p.m. until the required dose has been delivered (21). In the transformation experiments set up to isolate foci, C3H10T1/2 cells were irradiated with 5 Gy X-rays or 1 Gy {alpha}-particles, which resulted in similar levels of survival and transformation (Table IIGo).


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Table II. Survival and transformation frequencies
 
Stocks of C3H10T1/2 cells (passage 12) were removed from liquid nitrogen storage 1 week prior to irradiation. This allowed sufficient time for recovery and growth of cells and one subculture of the cells in the week preceding irradiation. One day prior to irradiation the cells were subcultured into 25 cm2 culture flasks for X-irradiation or Hostaphan dishes for {alpha}-particle irradiation, at cell numbers aimed to produce ~60% confluence on the day of irradiation.

Tumorigenicity studies
The tumour studies were carried out at the University of St Andrews (St Andrews, UK). C3H female mice (4–5 weeks old) received a whole body radiation dose of 5 Gy {gamma}-rays the day before injection of the focus or control C3H10T1/2 cells to be tested. Four million cells were injected s.c. per mouse and each cell line was tested in four to six mice. The site of injection was examined weekly for tumour formation for a maximum of 35 weeks and the sizes of any tumours recorded by palpating the tumour and comparing with graded sized ball bearings (22). The maximum tumour diameter reached before excision was ~9.5 mm. Average tumour sizes as a function of time after injection were calculated for each focus by averaging the results from the number of mice injected. When a tumour was excised the tumour diameter was measured in millimetres and kept in the calculations to estimate the average tumour size. The calculations for tumour growth for the partially tumorigenic foci included only tumour-bearing mice. Foci were termed `fully tumorigenic' when the cells produced tumours in all mice injected, while the term `partially tumorigenic' was applied to foci which did not produce tumours in all mice but did so in at least one.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The tumorigenicity data of the X-ray- and {alpha}-particle-induced foci are presented in Table IIIGo. It is evident from Table IIIGo that for both type I and type II/III foci the {alpha}-particle-induced foci were less tumorigenic than the X-ray-induced equivalents, with an average of 40% of {alpha}-particle-induced foci tumorigenic compared with ~85% of X-ray-induced foci. Tumour growth data are illustrated in Figures 1–3GoGoGo. Figure 1Go illustrates the growth of tumours induced by the transformed foci irrespective of whether the foci were fully or partially tumorigenic. Figures 2 and 3GoGo present the growth of tumours produced by fully and partially tumorigenic foci, respectively.


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Table III. Tumorigenicity data of X-ray- and {alpha}-particle-induced C3H10T1/2 foci
 


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Fig. 1. Growth of tumours (n) produced by {alpha}-particle-induced type II/III foci ({blacksquare}, n = 49) and type I foci ({blacktriangledown}, n = 20) and by X-ray-induced type II/III foci ({square}, n = 77) and type I foci ({triangledown}, n = 27).

 


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Fig. 2. Growth of tumours (n) produced by fully tumorigenic {alpha}-particle-induced type II/III foci ({blacksquare}, n = 26) and type I foci ({blacktriangledown}, n = 8) and by X-ray-induced type II/III foci ({square}, n = 56) and type I foci ({triangledown}, n = 16).

 


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Fig. 3. Growth of tumours (n) produced by partially tumorigenic {alpha}-particle-induced type II/III foci ({blacksquare}, n = 23) and type I foci ({blacktriangledown}, n = 12) and by X-ray-induced type II/III foci ({square}, n = 21) and type I foci ({triangledown}, n = 11).

 
Figure 1Go illustrates that both type II/III and type I X-ray-induced foci produced tumours which appear to have shorter latency periods than the {alpha}-particle-induced equivalents, although the difference is not substantial and once the tumours were evident they increased in size at similar rates. For both types of radiation the tumours produced by type II/III foci were evident before tumours produced by type I foci.

Figure 2Go presents the growth of tumours induced by fully tumorigenic foci only. As in Figure 1Go, the X-ray-induced type II/III foci produced tumours which had shorter latency periods but grew at similar rates to the tumours produced by the corresponding {alpha}-particle-induced foci. Irrespective of radiation type, tumours produced by type II/III transformed foci appeared earlier than those produced by type I foci.

Figure 3Go illustrates the growth of tumours produced by partially tumorigenic foci only, i.e. foci which produced at least one tumour but did not produce tumours in all mice injected. The growth was calculated as mean tumour size based on the number of tumour-bearing mice only. Tumours produced by the X-ray-induced type I foci had shorter latency periods compared with the {alpha}-particle-induced equivalents, although the growth curves were similar.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is evident from Table IIIGo that a greater proportion of the X-ray-induced foci were tumorigenic while the majority of the {alpha}-particle-induced foci were not tumorigenic for all focus types. Most of the tumorigenic foci produced the first tumour within 10 weeks of injection of the focus cells into the C3H mice and many of the X-ray-induced type II/III foci had produced all tumours within 20 weeks of injection of focus cells into the mice. {alpha}-Particle-induced type II/III foci required more time (up to 32 weeks) for all tumours to become apparent. All tumours from the X-ray-induced type I foci were evident within 12 weeks of injection of the focus cells, while those from the corresponding {alpha}-particle-induced foci took twice as long.

The tumorigenicity data for C3H10T1/2 foci obtained by other authors (Table IGo) using a similar process as used in this publication varied from 0 to 100% for both type II and type III foci. High proportions of X-ray-induced foci have been reported to be tumorigenic (48). Here, data for the X-ray-induced type II/III foci show tumorigenicity of 85%, while the corresponding tumorigenicity for the {alpha}-particle-induced type II/III foci is considerably less at 42%. Table IIGo illustrates the similar levels of survival and transformation for 1 Gy {alpha}-particles versus 5 Gy X-rays, thus in order to obtain similar transformation frequencies a 5-fold difference in dose is required. However, the tumorigenicity results from foci isolated under these similar conditions indicate only a 2-fold difference between X-rays and {alpha}-particles. This has implications for radiation protection studies where in vitro assays such as the C3H10T1/2 cell transformation assay may indicate a higher biological effectiveness of high LET radiation compared with low LET radiation than studies in vivo.

Type I foci were originally reported as being non-tumorigenic (1,2) and a number of reports have confirmed these findings (for example refs 4,5,9), although sample numbers are small. Aside from the possibility of misclassification, the relatively high tumour incidence of the type I foci presented here may be explained if it is assumed that type I foci are precursors of type II and type III and that some partially transformed cells became transformed in the time taken for the cells to be isolated from the focus, subcultured and the cell number increased for subsequent injection into the C3H mice. However, this is unlikely as, in the course of this work, non-tumorigenic type I foci and unirradiated parent C3H10T1/2 cells have been expanded for several generations in culture and subsequently tested for tumorigenicity at 20, 30 and 40 passages (up to ~20 weeks in culture) after focus cell isolation. No tumours developed. Although this does not preclude type I foci being precursors of type II and III foci, it suggests that additional steps are required other than extended passage in culture for this to be the case. These `steps' could be changes in the levels of oncogenes or tumour suppressor genes or alternatively an epigenetic alteration, or any combination of these. The identity of the possible changes involved in C3H10T1/2 transformed cells compared with normal cells is not clear (for example refs 23–25).

There is a wide range of evidence that many of the effects of radiation are due to its ability to damage DNA, for example by inducing double-strand breaks (studies summarized in ref. 26). High LET radiation damage is widely considered to be qualitatively different to that of low LET radiation damage. One explanation for these differences in tumorigenicity is the pattern of energy deposition of the various radiation types. High LET radiation, such as {alpha}-particles, deposit their energy in a much more localized area than low LET radiation such as X-rays. Even when the same amount and types of damage are induced, the cell repair mechanism is more able to correct damage if it is well spaced so as to prevent interaction of two or more damaged sites (27). The proximity of damage induced by high LET radiation is likely to increase the probability of misrepair (27). At the DNA level localized clusters of damage, including complex double strand-breaks, are more likely to be induced by high LET radiation and increase the probability of misrepair (28,29). Several studies report that high LET radiation damage is not as efficiently repaired as that produced by low LET radiation (30,31). The misrepair of DNA damage, such as strand breaks, may have several consequences, including lethality or transformation, for the affected cell. There is increasing evidence that high LET radiation is very efficient in inducing instability in the genome and may take several generations to manifest itself. Two studies in particular report genome instability induced by {alpha}-particles but not by X-rays (33,34). Other studies, using a hamster/human hybrid cell line, show chromosomal instability induced by X-rays (for example ref. 35). Studies on induction of sister chromatid exchanges in lymphocytes suggest that the {alpha}-particle-induced DNA lesions are retained longer than those induced by X-rays. These lesions are then carried into the S phase of the cell cycle where they can be converted into sister chromatid exchanges (33). The kinetics of induction of sister chromatid exchanges by {alpha}-particles and X-rays bear certain similarities to the induction of transformation (35,36).

One explanation for the tumorigenicity data presented in this work is the induction of chromosomal instability by high LET radiation and also, to a lesser extent, by the low LET radiation. Transformation may be regarded as a step on the route to carcinogenesis of a normal cell. Although the radiations induced sufficient damage to induce transformation in the parent cells from which the cell lines tested for tumorigenicity were developed, this damage may not have been sufficient in all cases to produce tumorigenic transformants. {alpha}-Particle-induced foci were less tumorigenic than the X-ray-induced foci, under the conditions of this assay, and where tumours developed they appeared later than those produced by the X-ray-induced foci. The {alpha}-particles may have induced instability in the focus parent cell genome at the time of irradiation which takes a considerable period of time to manifest itself. This instability may manifest itself as aberrations before or after focus formation leading to tumorigenicity or the aberrations may prove lethal to the focus cells, making them non-viable and thus resulting in an increase in cell death and a lower tumour incidence. It is thus possible that, given a longer period of incubation in vivo, the {alpha}-particle-induced foci would reach the same level of tumorigenicity as the X-ray-induced foci.

One clear conclusion from these studies is that it is very important to define the criteria to be used in the transformation assay, in particular the criteria adopted to distinguish positively transformed foci. This is especially important to allow comparison of data such as transformation frequencies between laboratories. Another important aspect of this assay is to identify and clarify the true parameter of interest, for example one of the most important aspects of the foci identified in the C3H10T1/2 transformation assay is their tumorigenic potential, i.e. how the observation in vitro relates to the in vivo situation. While it is not feasible to examine all foci for tumorigenicity, it is imperative that individual laboratories using the C3H10T1/2 assay examine the tumorigenicity of some foci, deemed to be positively transformed, in order to optimize the criteria within the laboratory for the assessment of truly transformed foci. The data presented here may indicate an underestimation of the absolute transformation frequencies using this assay where some type I foci should possibly be included in the type II/III focus category, based on our tumorigenicity results. This affects interpretation of absolute transformation frequencies more so than comparison studies which have been done, for example assessing the relative biological effectiveness of high LET to low LET radiation within a laboratory.

Another important observation was that the relationship between transformation and tumorigenicity was found to be dependent on radiation type. This factor also needs to be considered when optimizing the transformation assay and when comparing the effectiveness of different radiation types.


    Notes
 
4 To whom correspondence should be addressed at present address: Department of Medical Oncology, Paterson Institute of Cancer Research, Wilmslow Road, Withington, Manchester M20 4BX, UK Email: mlehane{at}picr.man.ac.uk Back


    Acknowledgments
 
The assessment of positively and non-positively transformed foci was carried out in conjunction with a collaborative project on transformation by low doses of X-rays, partially funded by the Commission of the European Communities contract F13P-CT920043. Collaborators were D.Bettega et al. (Universita degli Studi di Milano, Italy), D.Frankenberg et al. (Georg-August-Universität Göttingen, Germany), L.Hieber et al. (GSF-Forschungzentrum, München, Germany), W.F.Morgan et al. (AEA Technology, Harwell, UK) and A.Saran et al. (Ente per le Nuove Tecnologie, Rome, Italy). {alpha}-Particle irradiations were performed by D.Stevens et al. (MRC Radiobiology Unit, Harwell, UK). These studies were jointly funded by Nuclear Electric Ltd, Scottish Nuclear Ltd and Magnox Electric plc. This paper is published by permission of Nuclear Electric Ltd.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 2, 1997; revised September 29, 1998; accepted September 29, 1998.