Skip to main content
NCBI home page
Toxicological Research logoLink to Toxicological Research
. 2008 Mar 1;24(1):37–44. doi: 10.5487/TR.2008.24.1.037

Comparison of In Vitro Cell Transformation Assay Using Murine Fibroblasts and Human Keratinocytes

Jun-Ho Ahn 16, Sue Nie Park 26, Yung-Na Yum 26, Ji-Young Kim 36, Michael Lee 16,✉
PMCID: PMC7006257  PMID: 32038775

Abstract

The in vitro cell transformation assays (CTA) were performed using BALB/3T3 murine fibroblasts and HaCaT human keratinocytes in order to evaluate concordance between both in vitro CTAs and carcinogenicity with compounds differing in their genotoxic and carcinogenic potential. Six test articles were evaluated, two each from three classes of compounds: genotoxic carcinogens (2-amino-5-nitrophenol and 4-nitroquinoline-N-oxide), genotoxic noncarcinogens (8-hydroxyquinoline and benzyl alcohol), and nongenotoxic carcinogens (methyl carbamate and N-nitrosodiphenylamine). Any foci of size ≥ 2 mm regardless of invasiveness and piling was scored as positive in CTA with BALB/3T3. As expected, four carcinogens regardless of their genotoxicity had positive outcomes in two-stage CTA using BALB/3T3 cells. However, of the two genotoxic noncarcinogens, benzyl alcohol was positive CTA finding. We concluded that, of the 6 chemicals tested, the sensitivity for BALB/3T3 system was reasonably high, being 100%. The respective specificity for BALB/3T3 assay was 50%. We also investigated the correlation between results of BALB/3T3 assay and results from HaCaT assay in order to develop a reliable human cell transformation assay. However, evaluation of staining at later time points beyond the confluency stage did not yield further assessable data because most of HaCaT cells were detached after 2~3 days of confluency. Thus, after test article treatment, HaCaT cells were split before massive cell death began. In this modified protocol for this HaCaT system, growing attached colonies were counted instead of transformed foci 3 weeks since last subculture. Compared to BALB/3T3 assay, HaCaT assay showed moderate low sensitivity and high specificity. Despite these differences in specificity and sensitivity, both cell systems did exhibit same good concordance between in vitro CTA and rodent carcinogenicity findings (overall 83% concordant results). At present the major weakness of these in vitro CTA is lack of validation for regulatory acceptance and use. Thus, more controlled studies will be needed in order to be better able to assess and quantitatively estimate in vitro CTA data.

Key words: Carcinogenicity, In vitro cell transformation assay, Murine fibroblasts, BALB/3T3, Human keratinocytes, HaCaT

References

  1. Boukamp P, Peter W, Pascheberg U, Altmeier S, Fasching C, Stanbridge EJ, Fusenig NE. Stepwise progression in human skin carcinogenesis in vitro involves mutational inactivation of p53, ras-H oncogene activation and additional chromosome loss. Oncogene. 1995;11:961–969. [PubMed] [Google Scholar]
  2. Boukamp P, Petrussevka RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 1988;106:761–771. doi: 10.1083/jcb.106.3.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boukamp P, Popp S, Altmeyer S, Hulsen A, Fasching C, Cremer T, Fusenig NE. Sustained non-tumorigenic phenotype correlates with a largely stable chromosome content during long-term culture of the human keratinocyte cell line HaCaT. Genes Chromosomes Cancer. 1997;19:201–214. doi: 10.1002/(SICI)1098-2264(199708)19:4<201::AID-GCC1>3.0.CO;2-0. [DOI] [PubMed] [Google Scholar]
  4. Combes R, Balls M, Curren R, Fischbach M, Fusenig N, Kirkland D, Lasne C, Landolph J, LeBoeuf R, Marquardt H, et al. Cell transformation assays as predictors of human carcinogenicity. The report and recommendations of ECVAM Wokshop 39. ATLA. 1999;27:745–767. doi: 10.1177/026119299902700505. [DOI] [PubMed] [Google Scholar]
  5. Fusenig NE, Boukamp P. Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin kerati-nocyes. Mol. Carcinog. 1998;23:144–158. doi: 10.1002/(SICI)1098-2744(199811)23:3<144::AID-MC3>3.0.CO;2-U. [DOI] [PubMed] [Google Scholar]
  6. IARC/NCI/EPA Working Group 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. 1985;45:2395–2399. [Google Scholar]
  7. Kajiwara Y, Ajimi S. Verification of the BALB/c 3T3 cell transformation assay after improvement by using an ITES-medium. Toxicol. In Vitro. 2003;17:489–496. doi: 10.1016/S0887-2333(03)00050-X. [DOI] [PubMed] [Google Scholar]
  8. Kakunaga T. A quantitative system for assay of malignant transformation by chemical carcinogens using a clone derived from Balb 3T3. Int. J. Cancer. 1973;12:463–473. doi: 10.1002/ijc.2910120217. [DOI] [PubMed] [Google Scholar]
  9. Kakunaga T, Yamasaki HE. Transformation assay of established cell lines: mechanisms and application. IARC, Lyon: IARC Scientific Publication No. 67; 1985. [PubMed] [Google Scholar]
  10. Keshava N. Tumorigenicity of morphologically distinct transformed foci induced by 3-methylcholanthrene in BALB/C-3T3 cells. Mutat. Res. 2000;447:281–286. doi: 10.1016/S0027-5107(99)00216-X. [DOI] [PubMed] [Google Scholar]
  11. Kirkland D, Aardema M, Henderson L, Muller L. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity. Mutat. Res. 2005;584:1–256. doi: 10.1016/j.mrgentox.2005.02.004. [DOI] [PubMed] [Google Scholar]
  12. Kirkland D, Aardema M, Muller L, Hayashi M. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens II. Further analysis of mammalian cell results, relative predictivity and tumour profiles. Mutat. Res. 2006;608:29–42. doi: 10.1016/j.mrgentox.2006.04.017. [DOI] [PubMed] [Google Scholar]
  13. Landolph, J.R. (1985). Chemical transformation in C3H10T 1/2 CC118 mouse embryo fibroblasts: historical background, assessment of the transformation assay, and evolution and optimization of the transformation assay protocol in Transformation Assay of Established Cell Lines: Mechanisms and Application IARC Scientific Publications No. 67 (Kakunaga, T. and Yamasaki, H. Eds.). IARC, Lyon, pp. 185–203. [PubMed]
  14. LeBoeuf RA, Kerckaert KA, Aardema MJ, Isfort RJ. Use of Syrian hamster embryo and Balb/c 3T3 cell transformation for assessing the carcinogenicity potential of chemicals in The Use of Short- and Medium-term Tests for Carcinogenic Hazard Evaluation. In: McGregor JM R, Venitt S, editors. IARC Scientific Publications No. 146. Lyon: IARC; 1999. pp. 409–425. [PubMed] [Google Scholar]
  15. Lee M, Kwon J, Choi J-H, Hong M-Y, Kim E-J, Han J, Chung M-K, Han SS. Comparison of L5178Y tk+/ mouse lymphoma assay and in vitro chromosome aberration test. J. Toxicol. Pub. Health. 2002;18:129–138. [Google Scholar]
  16. Lee JK, Lee EH, Lee SH. Comparison of sensitivity between Balb/c 3T3 cell and HaCaT cell by NRU assay to predict skin phototoxicity potential. J. Toxicol. Pub. Health. 2002;18:227–232. [Google Scholar]
  17. Matthews EJ. Transformation of Balb/c 3T3 cells. I. Investigation of experimental parameters that influence detection of spontaneous transformation. Environ. Health Perspect. 1993;101:277–291. doi: 10.1289/ehp.93101s2277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Matthews EJ. Transformation of Balb/c 3T3 cells. II. Investigation of experimental parameters that influence detection of benzo[a]pyrene-induced transformation. Environ. Health Perspect. 1993;101:293–310. doi: 10.1289/ehp.93101s2293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Matthews EJ. Transformation of Balb/c 3T3 cells. III. Development of a co-culture clonal survival assay for quantification of chemical cytotoxicity in high-density cell cultures. Environ. Health Perspect. 1993;101:311–318. doi: 10.1289/ehp.93101s2311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Matthews EJ, Spalding JW, Tennant RW. Transformation of BALB/c 3T3 cells. IV. Rank-ordered potency of 24 chemical responses detected in a sensitive new assay procedure. Environ. Health Perspect. 1993;101:347–482. doi: 10.1289/ehp.93101s2319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Matthews EJ, Spalding JW, Tennant RW. Transformation of BALB/c 3T3 cells. V. Transformation responses of 168 chemicals compared with mutagenicity in Salmonella and carcinogenicity in rodent bioassays. Environ. Health Perspect. 1993;101:347–482. doi: 10.1289/ehp.93101s2347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Maurici D, Aardema M, Corvi R, Kleber M, Krul C, Laurent C, Loprieno N, Pasanen M, Pfuhler S, Phillips B, Sabbioni E, Sanner T, Vanparys P. Genotoxicity and mutagenicity. Altem. Lab. Anim. 2005;33:117–130. doi: 10.1177/026119290503301s13. [DOI] [PubMed] [Google Scholar]
  23. Muller L, Miltenburger HG, Marquardt H, Madie S. In Current Issues in Genetic Toxicology. Munich, Germany: Medizin-Verlag; 1993. The significance of in vitro cell transformation assays for routine testing; pp. 102–113. [Google Scholar]
  24. Nakajima M, Shimada S, Nagai M, Mizuhashi F, Sugiyama C, Masuda S, Hayashi M, Kinae N. 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone [MX] shows initiating and promoting activities in a two-stage BALB/c 3T3 cell transformation assay. Mutagenesis. 2005;20:375–379. doi: 10.1093/mutage/gei050. [DOI] [PubMed] [Google Scholar]
  25. OECD . OECD Guidelines for the Testing of Chemicals (May 12, 1981) TG No. 451 ‘Carcinogenicity studies’. 1981. [Google Scholar]
  26. OECD . Series on testing and assessment (August 13, 2007) Number 31 Detailed review paper on cell transformation assays for detection of chemical carcinogens. 2007. [DOI] [PubMed] [Google Scholar]
  27. Piao L, Shin S, Yang K-J, Park J, Shin E, Li Y, Park KA, Byun HS, Won M, Lee CJ, Hur GM, Seok JH, Kim JD, Kim CD, Lee J-H, Park J. Calcium-induced human keratinocyte (HaCaT) differentiation requires protein kinase B activation in phosphatidyli-nositol 2-kinase-dependent manner. J. Toxicol. Pub. Health. 2006;22:283–291. [Google Scholar]
  28. Sakai A, Sato M. Improvement of carcinogen identification in BALB/3T3 cell transformation by application of a 2-stage method. Mutat. Res. 1989;214:285–296. doi: 10.1016/0027-5107(89)90172-3. [DOI] [PubMed] [Google Scholar]
  29. Tsuchiya T, Umeda M. Improvement in the efficiency of the in vitro transformation assay method using BALB/3T3 A31-1-1 cells. Carcinogenesis. 1995;16:1887–1894. doi: 10.1093/carcin/16.8.1887. [DOI] [PubMed] [Google Scholar]
  30. Tsuchiya T, Umeda M, Nishiyama H, Yoshimura I, Ajimi S, Asakura M, Baba H, Dewa Y, Ebe Y, Fushiwaki Y, et al. An interlaboratory validation study of the improved transformation assay employing Balb/c 3T3 cells: Results of a collaborative study on the two-stage cell transformation assay by the Non-genotoxic Carcinogen Study Group. ATLA. 1999;27:685–702. doi: 10.1177/026119299902700409. [DOI] [PubMed] [Google Scholar]

Articles from Toxicological Research are provided here courtesy of Korean Society of Toxicology

RESOURCES