BAT Science - Cancer

Cancer is a leading cause of death worldwide.  From a total of 58 million deaths worldwide in 2005, cancer accounts for 7.6 million (or 13%) of all deaths (WHO figures [ 1  ]). Lung cancer is by far the biggest cause of death, accounting for 22% of all cancer deaths in the UK in 2004.

The link between cancer and certain environmental and lifestyle exposures has been observed and reported for hundreds of years. For example, in 1761, the English physician John Hill noted the high rates of nasal cancer among snuff users [4].  Percival Pott, a London surgeon, observed the high incidence of skin cancer of the scrotum of former chimney sweeps in a report published in 1775 [5].  Several other reports of occupational exposures to tar or tarry substances causing cutaneous cancer led to the official recognition of this association in Britain in 1907 [6].

Many of the early mechanistic studies on chemical carcinogenesis were performed on animals.  However, over the past forty years there has been a steady increase in the use of alternative methods to animal testing in this and other areas of biological research [7].  Pressure from the public and a desire amongst many scientists to adopt Russell and Burch’s ‘three Rs’ (replacement, refinement and reduction) of animal use wherever possible has been a contributory factor to this drive [8].  In vitro models can potentially serve as replacements to animal models, and have increased in use in parallel with advances in cell culture techniques. 

As in vitro models have become more sophisticated, due to advances in computer science and cellular and molecular biology [9], they are being applied more widely to mechanistic studies, and have contributed largely to the current knowledge of the origins of cancer. 

In vitro models to assess carcinogenic potential

In vitro assays have been developed as screens of cytotoxicity, mutagenicity and carcinogenicity. Genotoxicity test batteries have become a standard tool for identifying chemicals that may have potential carcinogenic risk to humans [10]. It is now apparent, however, that the use of genotoxicity batteries for assessing carcinogenic potential has limitations including an overall low specificity and a limited ability to detect carcinogens acting via 'non-genotoxic' mechanisms. This has led to the development of in vitro cell transformation models that measure a chemical's ability to induce preneoplastic (e.g. anchorage independent growth in soft agar) or neoplastic endpoints (e.g. tumourigenesis when transplanted into the flanks of nude mice) regardless of the mechanism(s) [11].

The recent Organisation for Economic Cooperation and Development (OECD) recommendations for the use of cell transformation assay systems in the testing of chemical carcinogens [12] give further weight to the development and application of these assays to the testing of cigarette smoke.  While the SHE cell transformation assay is becoming the method of choice for the study of the early stages in cell transformation (i.e. morphological transformation), an anchorage-independence assay may provide additional information on the later stages of cell transformation (e.g. immortalisation, cell migration).

In this section we describe the development and application of a number of in vitro models for use in testing of the carcinogenic effects of cigarette smoke and its constituents.

Whole smoke induces oxidative DNA damage in NCI-H292 lung epithelial cells (Comet assay).

DNA damage is considered to play an important role in carcinogenesis and has the potential to cause DNA mutations [13,14]. Reactive oxygen species (ROS) are of primary importance as they are thought to cause damage to lipids, proteins and DNA. ROS are produced either endogenously by cellular metabolism, or through exogenous exposure to environmental mutagens such as ultraviolet light [15], ionising radiation [16], and tobacco smoke [13,17,18]. ROS induce a wide range of DNA lesions, including 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodGuo), one of the most commonly used biomarkers of DNA oxidation in vitro and in vivo [19].

The single-cell gel electrophoresis assay (comet assay) is a rapid and sensitive microscopic method for the measurement of DNA damage induction and repair in mammalian cells. Coupled with the use of specific glycosylases and/or endonucleases, the comet assay has provided an easy and sensitive means to measure oxidative DNA-base modifications in vitro.

To investigate the potential effects of cigarette smoke on DNA, we have employed the in vitro comet assay using the lesion specific enzyme formamidopyrimidine N-glycosylase (FPG) [20], in conjunction with a novel whole smoke exposure platform [21].

The images above show an example of representative data from the in vitro comet assay. Lung epithelial H292 cells were exposed to sterile air (control) or whole cigarette smoke (minimal and extensive damage) at different concentrations. The extent of DNA damage is measured by the size and intensity of the tail produced from a single nucleous following electrophoreisis [20].

Our results find that oxidative DNA damage occurred in NCI-H292 cells following exposure to whole smoke. When cells were allowed to recover, the levels of non-oxidative DNA damage were reduced in a time-dependent fashion, and a clear FPG-associated oxidative dose response was revealed after a 16 hour recovery period. Our data further suggests that single-strand DNA breaks repair more quickly than oxidative lesions [22].

With the combination of the comet assay and the whole smoke exposure system we now potentially have a sensitive and versatile tool for the measurement of cigarette smoke induced oxidative DNA damage [22].

TPM acts as both initiator and promoter in Syrian hamster embryo cells (SHE cell transformation assay).

Morphological cell transformation is the term used to describe the process by which normal cultured cells are altered in both their behaviour and growth characteristics. These alterations can manifest themselves phenotypically as changes in cell morphology and disorganised growth patterns, and in some cases, loss of anchorage independence [23]. The induction of morphological transformation in Syrian hamster embryo (SHE) cells was first reported by Berwald and Sachs in 1963 [24]. This endpoint is more general than those induced in traditional in vitro genotoxicity assays such as the Salmonella typhimurium mutation assay (Ames assay) and mouse lymphoma assay which detect mutational events, and the in vitro cytogenetics assays (e.g.  micronucleus assay) which have structural and/or numerical chromosomal aberrations as their endpoints. This property may be of high predictive value, as carcinogenesis is a multistage process, resulting from a series of individual events which contribute to the eventual transformed phenotype. Changes in this endpoint may be indicative of the early stages in the carcinogenic process which may not be easily identifiable in other assay systems.

The SHE cell transformation assay is the most established transformation assay utilising primary cells, in terms of predicting known rodent carcinogens, and currently the most sensitive and specific [25]. The SHE cell transformation assay is often capable of detecting promoters and carcinogens that are not picked up by tests for genotoxicity (e.g. clofibrate and diethylhexylphthalate), and is therefore considered a promising in vitro test for potential non-genotoxic carcinogens. The endpoint of the assay, morphological transformation (MT), presents as a colony phenotype which is assessed by microscopic examination. MT colonies show disorganised growth patterns and piling up of cells, compared to normal colonies which display contact inhibition and ordered morphology.

Development of a two-stage SHE cell transformation assay

We developed a protocol for the pH 6.7 SHE transformation assay which separates the cell transformation process into two phases, potentially analogous to initiation and promotion in vivo. This allows chemicals found to be positive in the traditional SHE cell transformation assay to be further classified as initiators or promoters.

Following validation with known initiators, benzo(a)pyrene (B(a)P) and N-methyl-N-nitro-N-nitrosoguanidine (NNN) and promoters, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phenobarbitone, the two-stage model was applied to cigarette smoke particulates (SP).

The two-stage model was found to work successfully. The results of this study reported that (SP - smoke particulate) TPM acts at both the initiation and promotion stages of SHE cell transformation, and this provides a foundation to examine the interactions of tobacco smoke in the SHE cell transformation assay.

Molecular Markers of SHE cell transformation

Since the scoring of morphological transformation (MT) is both subjective and lacking in a mechanistic basis, leading to some criticism of the assay [26], we investigated the use of a molecular endpoint to identify MT cells. Relative mRNA expression of a panel of 12 genes considered to be involved in cell transformation and/or carcinogenesis was compared between normal and MT colonies using real-time polymerase chain reaction (PCR).

 

Gene	Mechanism	Fold-change in gene expression ± SD (n=3)
Cyclin D1 (Ccnd1)	Cell cycle	25.8±14.2
Cyclo-oxygenase 2 (Cox2)	Inflammation	6.5±1.2
Insulin-like growth factor (Igf2)	Embryogenesis	0.1±0
B cell leukemia / lymphoma 2 (Bcl2)	Apoptosis	2.3±0.7
Matrix metalloproteinase 2 (Mmp2)	Vascular invasion	2.8±0.3
Vascular endothelial growth factor (Vegf)	Angiogenesis	2±0.6
Tropomyosin-1 (Trop1)	Metastasis	2±0.3
Bcl-2 associated x protein (Bax)	Apoptosis	1.5±0.2
c-Myc (myc)	Proliferation	1.7±0.6
Survivin (Svn)	Apoptosis inhibitor	0.4±0.1
Osteopontin (Opn)	Invasion and metastasis	2.6±0.3
p53 (Tp53)	Tumour suppressor	1.4±0.3

Results showed that gene expression analysis can be used to identify potential genetic markers of MT in the SHE cell transformation assay. Cyclin D1, Cox2 and Igf2 are potentially suitable for further investigation as markers of MT and could be incorporated into an automated and objective endpoint for SHE cell transformation.

Anchorage-independent growth, a hallmark of cellular transformation, is induced by TPM (AIG assay).

As described above, the acquisition of MT in response to cigarette smoke has been characterised in a two-stage SHE cell transformation assay developed in GR&D [27].  However, there is an additional requirement to assess how cigarette smoke affects later stages in multistage cell transformation so that this can be incorporated into an overall assessment of carcinogenic potential.

One of the phenotypic changes associated with such later events of cell transformation following immortalisation is the gain of anchorage-independent growth (AIG). AIG is characterised by the ability of cells to grow in a semi-solid matrix such as agar or agarose.  Cigarette smoke and its constituents have previously been shown to induce anchorage-independent growth (AIG) in a number of cell lines including BEAS-2B cells, an SV40-immortalised normal human bronchial epithelial cell line that has been used extensively in carcinogenicity studies [28,29,30]. We are currently working on developing an AIG assay using cigarette smoke total particulate matter in BEAS-2B cells, and preliminary data show that TPM induces AIG in this cell line.

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1. http://www.who.int/en/
2. Doll, R., Peto, R. (1981).  The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. Journal of the National Cancer Institute. 66 (6): 1191-1308.
3. Peto, J. Cancer epidemiology in the last century and the next decade. (2001). Nature. 411 (6835): 390-395.
4. Hill, J. Cautions Against the Immoderate Use of Snuff. Founded on the Known Qualities of the Tobacco Plant; And the Effects it Must Produce when this Way Taken into the Body: And Enforced by Instances of Persons who have Perished Miserably of Diseases, Occasioned, or Rendered Incurable by its Use (R. Baldwin and J. Jackson, London, 1761).
5. Pott, P. The Chirurgical Works. Chirurgical Observations Relative to the Cataract, The Polypus of the Nose, The Cancer of the Scrotum, The Different Kinds of Ruptures, and The Mortification of the Toes and Feet Ch. III 60-68 (Hawes, W. Clarke, and R. Collins, London, 1775).
6. Henry, S. A. (1947).  Occupational cutaneous cancer attributable to certain chemicals in industry. British Medical Bulletin. 4: 389-401.
7. Knight, DJ, Breheny, D. (2002).  Alternatives to animal testing in the safety evaluation of products. Alternatives to Laboratory Animals: ATLA. 30 (1): 7-22.
8. Russell, W. M. S, Burch, R. L. (1959). The Principles of Humane Experimental Technique. London: Methuen & Co Ltd.
9. Bhogal, N., Grindon, C., Combes, R., Balls, M. (2005).  Toxicity testing: creating a revolution based on new technologies. Trends in Biotechnology. 23 (6): 299-307.
10. International Conference for Harmonisation (1999). Guidance for Industry. S2B Genotoxicity: A standard battery for the genotoxicity testing of pharmaceuticals.  1-12.
11. Freedman, V. H., Shin, S. I. (1974). Cellular tumorigenicity in nude mice: correlation with cell growth in semi-solid medium. Cell. 3 (4): 355-359.
12. OECD. OECD Environmental Health and Safety Publications Series on Testing and Assessment No. 31.  Detailed review paper on non-genotoxic carcinogens detection: The performance of in-vitro cell transformation assays (2001). Paris, Environment Directorate Organisation for Economic Co-coperation and Development.
13. Pourcelot, S., Faure, H., Firoozi, F., Ducros, V., Tripier, M., Hee, J., Cadet, J., Favier, A. (1999). Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and 5-(hydroxymethyl) uracil in smokers. Free Radical Research. 30 (3): 173-180.
14. Jenner, A., England, T. G., Aruoma, O. I., Halliwell, B. (1998). Measurement of oxidative DNA damage by gas chromatography-mass spectrometry: ethanethiol prevents artifactual generation of oxidized DNA bases. Biochemical Journal. 15 (331 Pt.2): 365-369.
15. Epe, B. (1991). Genotoxicity of singlet oxygen. Chemico-Biological Interactions. 80 (3): 239-260.
16. Frelon, S., Douki, T., Ravanat, J. L., Pouget, J. P., Tornabene, C., Cadet, J. (2000). High-performance liquid chromatography--tandem mass spectrometry measurement of radiation-induced base damage to isolated and cellular DNA. Chemical Research Toxicology. 13 (10): 1002-1010.
17. Flicker, T. M. and Green, S. A. (2001). Comparison of gas-phase free-radical populations in tobacco smoke and model systems by HPLC. Environment Health Perspectives. 109 (8): 765-771.
18. Valavanidis, A., Vlachogianni, T., Fiotakis, K. (2009). Tobacco smoke: involvement of reactive oxygen species and stable free radicals in mechanisms of oxidative damage, carcinogenesis and synergistic effects with other respirable particles. International Journal of Environmental Research and Public Health. 6 (2): 445-462.
19. Valavanidis, A., Vlachogianni, T., Fiotakis, C. (2009). 8-hydroxy-2' -deoxyguanosine (8-OHdG): A critical biomarker of oxidative stress and carcinogenesis. Journal of Environmental Science and Health. Part C. Environmental Carcinogenesis & Ecotoxicology Reviews. 27 (2): 120-139.
20. Dusinska, M., Collins, A. R. (1996). Detection of oxidised purines and UV-induced photoproducts in DNA of single cells, by inclusion of lesion-specific enzymes in the Comet Assay. Alternatives to Laboratory Animals: ATLA. 24 (3): 405-411. 
21. Phillips, J., Kluss, B., Richter, A., Massey, E. D. (2005). Exposure of Bronchial Epithelial Cells to Whole Cigarette Smoke: Assessment of Cellular Responses.  Alternatives to Laboratory Animals: ATLA. 33 (3): 239-248.
22. Thorne, D., Wilson, J., Kumaravel, T. S., Massey, E.D., McEwan, M. (2009). Measurement of oxidative DNA damage induced by mainstream cigarette smoke in cultured NCI-H292 human pulmonary carcinoma cells. Mutation Research. 673(1):3-8. Link to manuscript abstract and citation. 
23. Combes, R., Balls, M., Curren, R., Fischbach, M., Fusenig, N., Kirkland, D., Lasne, C., Landolph, J., LeBoeuf, R., Marquardt, H., McCormick, J., Muller, L., Rivedal, E., Sabbioni, E., Tanaka, N., Vasseur, P., Yamasaki, H. (1999).  Cell transformation assays as predictors of human carcinogenicity.  The report and recommendations of ECVAM workshop 39.  Alternatives to Laboratory Animals: ATLA. 27 (5): 745-767.
24. Berwald, Y., Sachs, L. (1963).  In vitro cell transformation with chemical carcinogens.  Nature (London) 200: 1182-1184.
25. LeBoeuf, R. A., Kerckaert, G. A., Aardema, M. J., Gibson, D. P., Brauninger, R., Isfort, R. J.  (1996). The pH 6.7 Syrian hamster embryo cell transformation assay for assessing the carcinogenic potential of chemicals. Mutation Research. 356 (1): 85–127.
26. Committee on Mutagenicity on Chemicals in Food, Consumer Products and the Environment COM. ILSI/HESI research programme on alternative cancer models: results of Syrian hamster embryo cell transformation assay
    COM statement COM/02/S3 - April 2002.  
27. Breheny, D., Zhang, H., Massey, E. D. (2005). Application of a two-stage Syrian hamster embryo cell transformation assay to cigarette smoke particulate matter. Mutation Research. 572 (1-2): 45-57. Link to manuscript abstract and citation.  
28. Langenfeld, J., Lonardo, F., Kiyokawa, H., Passalaris, T., Ahn, M. J., Rusch, V., Dmitrovsky, E. (1996). Inhibited transformation of immortalized human bronchial epithelial cells by retinoic acid is linked to cyclin E down-regulation. Oncogene. 13 (9): 1983-1990.
29. van Agen, B., Maas, L. M., Zwingmann, I. H., Van Schooten, F. J., Kleinjans, J. C. (1997). B[a]P-DNA adduct formation and induction of human epithelial lung cell transformation. Environmental and Molecular Mutagenesis. 30 (3): 287-292.
30. Lemjabbar-Alaoui, H., Dasari, V., Sidhu, S. S., Mengistab, A., Finkbeiner, W., Gallup, M., Basbaum, C. (2006). Wnt and Hedgehog are critical mediators of cigarette smoke-induced lung cancer. PLoS One. 1: e93.