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Protocol no. 62
SCREENING SYSTEM OF PROMOTERS USING RAS TRANSFECTED BALB 3T3 CLONE (BHAS 42)

Tumour promoters can be detected by their ability to cause the initiated Bhas 42 cells to lose contact inhibition.

CONTACT

Dr Kiyoshi Sasaki
Laboratory of Cell Toxicology Department of Cell Biology
Hatano Research Institute Food and Drug Safety
Center 729-5 Ochiai Hadano Kanagawa 257 JAPAN
Tel: Japan - 463-82-4751 (ext 433) Fax: Japan - 463-82-9627

RATIONALE

BALB 3T3 cells transfected with a ras gene, produce cells initiated in two-stage transformation which can be applied to screening of promoters by using the two-stage transformation assay. Bhas 42 cells are v-Ha-ras-transfected BALB 3T3 cells cloned by co-transfection with pSV2-neo genes. The cells are sensitive to contact inhibition and undergo a drastic transformation on treatment with the known promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Bhas 42 cells are therefore suitable for screening promoters and antipromoters, and investigating the interaction between v-Ha-ras genes and specific promoters.

BASIC PROCEDURE

Bhas 42 cells and BALB 3T3 cells are cultured together and treated with the test compound for 2 weeks. The number of transformed foci is scored 6 weeks after plating and the transformation frequency expressed as the number of foci per plating efficiency of Bhas 42 cells.

CRITICAL ASSESSMENT

Chemical carcinogenesis is a complex multistep process that may be summarised as three basic steps, initiation, promotion and progression. The initiation step is the induction of genetic lesions in a small population of cells by a chemical carcinogen, and is not a dose dependent effect, one exposure to the carcinogen can cause initiation. This step is irreversible, but in itself is not sufficient to produce the fully developed tumour. The next step in the progress of the disease depends upon the clonal expansion of the initiated cells. This is brought about by any substance, which does not necessarily have to be a carcinogen itself, but promotes conditions that selectively favour the development of the initiated cells. This promotion step is reversible in as much as, preventing exposure to the promoter will halt the process. The definition of the transition from the promotion step to the progression step is less precise, but is generally the point at which the initiated cell population has become sufficiently expanded to be independent of the influence of the promoter. The rate of development of the disease during the progression step shows considerable variation in relation to several factors such as the cell type involved and the site in the body. For the detection of carcinogenic agents screening systems for initiators and promoters are required. Whilst there are a number of screens for initiators, screening for promoters is limited to chemically induced transformation systems such as those involving the chemical promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Various biochemical reactions are observed by treatment with TPA, such as activation of C-kinase, induction of DNA synthesis, expression of oncogenes, inhibition of EGF binding and induction of ornithine decarboxylase. Several chemicals (e.g. mezerein, dihydroteleocidin B, etc.) have similar effects and are referred to as TPA type promoters. On the other hand, some chemicals (e.g. hormones, growth factors, etc.) enhance tumour growth and transformation without these acts, and are referred to as non-TPA type promoters. The chemically induced transformation system is not an ideal screen for promoters as the results tend not to be reproducible. There are three main advantages in using the Bhas 42 transformation assay over the standard chemically-induced transformation system. Firstly, transformation of Bhas 42 cells is highly reproducible and is dependent on the concentration and duration of treatment with the tumour promoter. Secondly, in 3T3 and other cells, chemically-induced transformation can be influenced by the batches of serum used (Sasaki et al 1990ab). However, the transformation frequency of Bhas 42 cells is constant i.e. 20% of cells were shown to have transformed foci induced by treatment with TPA, in different serum batches. Thirdly, the Bhas 42 transformation system is easy to score objectively for foci, because most foci display a typical transformed morphology i.e. basophilic, multi-layered criss-cross, and fibroblastic phenotype. In chemically induced transformation assays, each transformed focus has an individual morphology, therefore there is potential for inaccuracies to arise due to subjectivity of the scoring. A further advantage of this system is, that by utilising established cell lines, it is possible to avoid the need for experimental animals and also, the complicated techniques involved in the isolation and establishment of primary cultures. In this transformation assay, co-culture of Bhas 42 cells with BALB 3T3 cells is necessary, since observation of morphological changes of Bhas 42 cells alone does not provide a quantitative measure of transformation. Also, it is not possible to observe drastic morphological changes by treatment with non-TPA type promoters such as sodium phenobarbital and sodium taurocholate. The co-culture method makes it possible to express the results quantitatively as transformation frequency, even for weak promoters. There is evidence to suggest that the BALB 3T3 cells inhibit transformation of the Bhas 42 cells, therefore the transformation frequency of Bhas 42 cells depends on the number of BALB 3T3 cells with which they are co-cultured. Although the Bhas 42 cell line was co-transfected with pSV2-neo and v-Ha-ras DNA, the Bhas 42 cells appear to be non-resistant to G-418. It appears that pSV2-neo DNA was deleted during proliferation, therefore the Bhas 42 cells should not be cultured in G-418 containing medium. The BALB 3T3 A31-1-1 cell line is recommended as the most appropriate cell line for co-culture in this assay, as no spontaneous transformed foci are observed following 6 weeks of culture. Whilst the transformation frequency of Bhas 42 cells is not affected by differences in serum batches, the plating efficiency of these cells and the BALB 3T3 cells are very sensitive to such differences. The character of both cell types can be altered by subculture methods, passage number and serum batches. It is possible to modify this protocol in order to get low and high transformation frequency in negative (solvent, test compounds and TPA treatment in BALB 3T3 cells alone, and solvent treatment in co-culture of Bhas 42 and BALB 3T3 cells) and positive (TPA treatment in co-culture of Bhas 42 and BALB 3T3 cells) controls, respectively. This can be achieved by altering such factors as the treatment dose, exposure period, number of cells plated out, culture period etc.

TEST STATUS

In house development. Studies using the Bhas 42 cell line are currently underway in other laboratories, these include a study of tumour and cell-cell communication, screening of antipromoters, and a biochemical study. As yet, no papers have been published on these investigations.

CHEMICALS TESTED

Anthralin 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT)
1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3)
Epidermal growth factor (EGF)
Fibroblast growth factor (FGF)
Insulin
Mezerein
Platelet derived growth factor (PDGF)
Sodium phenobarbital
Sodium taurocholate 12-O-tetradecanoylphorbol-13-acetate (TPA)

REFERENCES

  1. Dotto, G.P., Parada, L.F. & Weinberg, R.A. (1985) Specific growth response of ras-transformed embryo fibroblasts to tumour promoters. Nature, 318, 472-475.
  2. Hsiao, W.-L.W., Gattoni-Celli, S. & Weinstein, I.B. (1984) Oncogene-induced transformation of C3H 10T1/2 cells is enhanced by tumor promoters. Science, 226, 552-554.
  3. Hsiao, W.-L.W., Wu, T. & Weinstein, I.B. (1986) Oncogene-induced transformation of a rat embryo fibroblast cell line is enhanced by tumor promoters. Mol. Cell. Biol. 6, 1943-1950.
  4. IARC/NCI/EPA Working Group (1985) Cellular and molecular mechanisms of cell transformation and standardization of transformed assays of established cell lines for the prediction of carcinogenic chemicals: Overview and recommended protocols. Cancer Res. 45, 2395-2399.
  5. IARC/NCI/EPA Working Group (1985) Experimental protocols recommended by the working group. In: Transformation Assay of Established Cell Lines: Mechanisms and Application (IARC Scientific Publications No. 67), (eds. Kakunaga, T. & Yamasaki, H.), pp 207-219.
  6. Lyon: International Agency for Research on Cancer. Sasaki, K., Mizusawa, H., Ishidate, M. & Tanaka, N. (1990a) Transformation of ras transfected BALB 3T3 clone (Bhas 42) by promoters: Application for screening and specificity of promoters. Toxicology in Vitro, 4, 657-659.
  7. Sasaki, K., Mizusawa, H., Ishidate, M. & Tanaka, N. (1990b) Establishment of a highly reproducible transformation assay of a ras transfected BALB 3T3 clone by treatment with promoters. In: Antimutagenesis and Anticarcinogenesis Mechanisms II (Basic Life Sciences Vol. 52), (eds. Kuroda, Y., Shankel, D.M. & Waters, M.), pp 411-416. New York: Plenum Publishing Corp. Sasaki, K., Tanaka, N., Watanabe, M. & Yamada, M. (1991) Comparison of cytotoxic effects of chemicals in four different cell types. Toxic. in Vitro 5, 403-406.
  8. Sasaki, K. & Tanaka, N. (1991) Chemical contamination suspected in cytotoxic effects of incubators on BALB 3T3 cells. ATLA 19, 421-427.

IP-62 © October 1992