Improvement of the BALB/c-3T3 cell transformation assay: a tool for investigating cancer mechanisms and therapies

Doerte Poburski; René Thierbach

doi:10.1038/srep32966

journal article Open Access Sep 09, 2016

Improvement of the BALB/c-3T3 cell transformation assay: a tool for investigating cancer mechanisms and therapies

Doerte Poburski René Thierbach

Scientific Reports Vol. 6 No. 1 · Springer Science and Business Media LLC

View at Publisher Save 10.1038/srep32966

Abstract

AbstractThe identification of cancer preventive or therapeutic substances as well as carcinogenic risk assessment of chemicals is nowadays mostly dependent on animal studies. In vitro cell transformation assays mimic different stages of the in vivo neoplastic process and represent an excellent alternative to study carcinogenesis and therapeutic options. In the BALB/c-3T3 two-stage transformation assay cells are chemically transformed by treatment with MCA and TPA, along with the final Giemsa staining of morphological aberrant foci. In addition to the standard method we can show, that it is possible to apply other chemicals in parallel to identify potential preventive or therapeutic substances during the transformation process. Furthermore, we successfully combined the BALB/c cell transformation assay with several endpoint applications for protein analysis (immunoblot, subcellular fractionation and immunofluorescence) or energy parameter measurements (glucose and oxygen consumption) to elucidate cancer mechanisms in more detail. In our opinion the BALB/c cell transformation assay proves to be an excellent model to investigate alterations in key proteins or energy parameters during the different stages of transformation as well as therapeutic substances and their mode of action.

Topics

No keywords indexed for this article. Browse by subject →

References

42

[1]

Stewart, B. W. & Wild, C. P. World Cancer Report 2014. IARC (2014) http://www.who.int/mediacentre/factsheets/fs297/en/(08.02.2016).

[2]

Mascolo, M. G. et al. BALB/c 3T3 cell transformation assay for the prediction of carcinogenic potential of chemicals and environmental mixtures. Toxicol In Vitro 24, 1292–1300 (2010). 10.1016/j.tiv.2010.03.003

[3]

Perrin, S. Preclinical research: Make mouse studies work. Nature 507, 423–425 (2014). 10.1038/507423a

[4]

Barrett, J. C. & Ts’o, P. O. Evidence for the progressive nature of neoplastic transformation in vitro. Proc Natl Acad Sci USA 75, 3761–3765 (1978). 10.1073/pnas.75.8.3761

[5]

OECD. Detailed review paper on cell transformation assays for detection of chemical carcinogens (2007).

[6]

Sakai, A. BALB/c 3T3 cell transformation assays for the assessment of chemical carcinogenicity. Japanese Society for Alternatives to Animal Experiments 14, 367–373 (2007).

[7]

Berwald, Y. & Sachs, L. I. N. Vitro cell transformation with chemical carcinogens. Nature 200, 1182–1184 (1963). 10.1038/2001182a0

[8]

Schechtman, L. M. Rodent cell transformation assays-a brief historical perspective. Mutat Res 744, 3–7 (2012). 10.1016/j.mrgentox.2011.12.018

[9]

Sasaki, K. et al. Photo catalogue for the classification of foci in the BALB/c 3T3 cell transformation assay. Mutat Res 744, 42–53 (2012). 10.1016/j.mrgentox.2012.01.009

[10]

Vanparys, P. et al. Application of in vitro cell transformation assays in regulatory toxicology for pharmaceuticals, chemicals, food products and cosmetics. Mutat Res 744, 111–116 (2012). 10.1016/j.mrgentox.2012.02.001

[11]

Corvi, R. et al. ECVAM prevalidation study on in vitro cell transformation assays: general outline and conclusions of the study. Mutat Res 744, 12–19 (2012). 10.1016/j.mrgentox.2011.11.009

[12]

Kakunaga, T. A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from BALB-3T3. Int J Cancer 12, 463–473 (1973). 10.1002/ijc.2910120217

[13]

Isfort, R. J. & LeBoeuf, R. A. Application of in vitro cell transformation assays to predict the carcinogenic potential of chemicals. Mutat Res 365, 161–173 (1996). 10.1016/s0165-1110(96)90019-6

[14]

LeBoeuf, R. A. & Kerckaert, G. A. The induction of transformed-like morphology and enhanced growth in Syrian hamster embryo cells grown at acidic pH. Carcinogenesis 7, 1431–1440 (1986). 10.1093/carcin/7.9.1431

[15]

LeBoeuf, R. A. & Kerchaert, G. A. Enhanced morphological transformation of early passage Syrian hamster embryo cells cultured in medium with a reduced bicarbonate concentration and pH. Carcinogenesis 8, 689–697 (1987). 10.1093/carcin/8.5.689

[16]

Chouroulinkov, I. & Lasne, C. Two-stage (initiation-promotion) carcinogenesis in vivo and in vitro. Bull Cancer 65, 255–264 (1978).

[17]

Aaronson, S. A. & Todaro, G. J. Development of 3T3-like lines from Balb-c mouse embryo cultures: transformation susceptibility to SV40. J Cell Physiol 72, 141–148 (1968). 10.1002/jcp.1040720208

[18]

Kakunaga, T. & Crow, J. D. Cell variants showing differential susceptibility to ultraviolet light--induced transformation. Science 209, 505–507 (1980). 10.1126/science.7394516

[19]

Group, I. N. E. W. Cellular and Molecular Mechanisms of Cell Transformation and Standardization of Transformation Assays of Established Cell Lines for the Prediction of Carcinogenic Chemicals: Overview and Recommended Protocols. Cancer Res 45, 2395–2399 (1985).

[20]

Sakai, A. & Sato, M. Improvement of carcinogen identification in BALB/3T3 cell transformation by application of a 2-stage method. Mutat Res 214, 285–296 (1989). 10.1016/0027-5107(89)90172-3

[21]

Asada, S. et al. Detection of initiating as well as promoting activity of chemicals by a novel cell transformation assay using v-Ha-ras-transfected BALB/c 3T3 cells (Bhas 42 cells). Mutat Res 588, 7–21 (2005). 10.1016/j.mrgentox.2005.07.011

[22]

Sakai, A. et al. A Bhas 42 cell transformation assay on 98 chemicals: the characteristics and performance for the prediction of chemical carcinogenicity. Mutat Res 702, 100–122 (2010). 10.1016/j.mrgentox.2010.07.007

[23]

Sakai, A. et al. An international validation study of a Bhas 42 cell transformation assay for the prediction of chemical carcinogenicity. Mutat Res 725, 57–77 (2011). 10.1016/j.mrgentox.2011.07.006

[24]

Vaccari, M. et al. Identification of pathway-based toxicity in the BALB/c 3T3 cell model. Toxicol In Vitro 29, 1240–1253 (2015). 10.1016/j.tiv.2014.10.002

[25]

Sakai, A. Orthovanadate, an inhibitor of protein tyrosine phosphatases, acts more potently as a promoter than as an initiator in the BALB/3T3 cell transformation. Carcinogenesis 18, 1395–1399 (1997). 10.1093/carcin/18.7.1395

[26]

Umeda, M. Cell transformation assay using Balb/c 3T3 cells or Bhas 42 cells for the efficient detection of tumour promoters. Altern Lab Anim 32 Suppl 1B, 673–677 (2004). 10.1177/026119290403201s111

[27]

Ao, L. et al. Comparison of gene expression profiles in BALB/c 3T3 transformed foci exposed to tumor promoting agents. Toxicol In Vitro 24, 430–438 (2010). 10.1016/j.tiv.2009.10.006

[28]

Maeshima, H., Ohno, K., Tanaka-Azuma, Y., Nakano, S. & Yamada, T. Identification of tumor promotion marker genes for predicting tumor promoting potential of chemicals in BALB/c 3T3 cells. Toxicol In Vitro 23, 148–157 (2009). 10.1016/j.tiv.2008.10.005

[29]

McIntyre, A., Gibson, P. R. & Young, G. P. Butyrate production from dietary fibre and protection against large bowel cancer in a rat model. Gut 34, 386–391 (1993). 10.1136/gut.34.3.386

[30]

Heidor, R. et al. The chemopreventive activity of butyrate-containing structured lipids in experimental rat hepatocarcinogenesis. Mol Nutr Food Res (2015). 10.1002/mnfr.201500643

[31]

Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters.

M Castagna, Y Takai, K Kaibuchi et al.

Journal of Biological Chemistry 1982 10.1016/s0021-9258(18)34459-4

[32]

George, J. & Shukla, Y. Emptying of Intracellular Calcium Pool and Oxidative Stress Imbalance Are Associated with the Glyphosate-Induced Proliferation in Human Skin Keratinocytes HaCaT Cells. ISRN Dermatol 2013, 825180 (2013). 10.1155/2013/825180

[33]

Zhang, Y. J. et al. mTOR signaling pathway is a target for the treatment of colorectal cancer. Ann Surg Oncol 16, 2617–2628 (2009). 10.1245/s10434-009-0555-9

[34]

Lu, Q. et al. Expression and clinical significance of mammalian target of rapamycin/P70 ribosomal protein S6 kinase signaling pathway in human colorectal carcinoma tissue. Oncol Lett 10, 277–282 (2015). 10.3892/ol.2015.3228

[35]

Lee, S. Y., Jeong, E. K., Jeon, H. M., Kim, C. H. & Kang, H. S. Implication of necrosis-linked p53 aggregation in acquired apoptotic resistance to 5-FU in MCF-7 multicellular tumour spheroids. Oncol Rep 24, 73–79 (2010).

[36]

Holden, P. & Horton, W. A. Crude subcellular fractionation of cultured mammalian cell lines. BMC Res Notes 2, 243 (2009). 10.1186/1756-0500-2-243

[37]

Seshasai, S. R. et al. Diabetes mellitus, fasting glucose, and risk of cause-specific death. N Engl J Med 364, 829–841 (2011). 10.1056/nejmoa1008862

[38]

Sakai, A. & Fujiki, H. Promotion of BALB/3T3 cell transformation by the okadaic acid class of tumor promoters, okadaic acid and dinophysistoxin-1. Jpn J Cancer Res 82, 518–523 (1991). 10.1111/j.1349-7006.1991.tb01881.x

[39]

Kuroki, T. & Sasaki, K. Relationship between in-vitro cell transformation and in-vivo carcinogenesis based on available data on the effects of chemicals. IARC Sci Publ 67, 93–118 (1985).

[40]

Sasaki, K. et al. Recommended protocol for the BALB/c 3T3 cell transformation assay. Mutat Res 744, 30–35 (2012). 10.1016/j.mrgentox.2011.12.014

[41]

Tsuchiya, T. & Umeda, M. Improvement in the efficiency of the in vitro transformation assay method using BALB/3T3 A31-1-1 cells. Carcinogenesis 16, 1887–1894 (1995). 10.1093/carcin/16.8.1887

[42]

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

Marion M. Bradford

Analytical Biochemistry 1976 10.1016/0003-2697(76)90527-3

Metrics

26

Citations

42

References

Details

Published: Sep 09, 2016
Vol/Issue: 6(1)
License: View

Authors

Cite This Article

Doerte Poburski, René Thierbach (2016). Improvement of the BALB/c-3T3 cell transformation assay: a tool for investigating cancer mechanisms and therapies. Scientific Reports, 6(1). https://doi.org/10.1038/srep32966

Improvement of the BALB/c-3T3 cell transformation assay: a tool for investigating cancer mechanisms and therapies

You May Also Like