ELSEVIER Mutation Research 356 (1996) 1-3

Fundamental and Molecular Mechanisms of Mutagenesis

Preface

Hiroshi Yamasaki

01it of kfdlistage Carcinogenesis, International Agency for Research on Cancer, 150, cows Albert Thomas, 69372 Lyon, Cedex 08, France

In vitro cell-transformation systems have been developed so that at least a part of the carcinogenesis

process can be simulated with cultured cells, which are easier to manipulate than whole organisms (Berwald and Sachs, 1963). Since this is the only in vitro system in which ‘tumor’ production can be

utilized as an end-point, it occupies a unique place among numerous short-term tests for detecting po- tential carcinogens, most of which use gene or chro- mosomal mutations as endpoints. Because of their direct biological link to cancer, cell-transformation systems have also been extensively used to under- stand the molecular and cellular mechanisms of car-

cinogenesis. One important role played by cell-transformation

systems, but largely overlooked, is the identification

of oncogenes and tumor-suppressor genes. The dis- covery and cloning of the first activated human r-us

genes were achieved using NIH3T3 cell transforma- tion (Goldfarb et al., 1982; Shih and Weinberg,

1982; Pulciani et al., 1982). Syrian hamster embryo (SHE) cells have also been used as recipients of DNA transfection for identification of potential oncogenes (Higashi et al., 1990). A series of subse- quent studies with other types of cells in culture and cell transformation as the endpoint have revealed important roles of oncogenes and tumor-suppressor genes in carcinogenesis. These contributions to our molecular understanding of carcinogenesis were only possible because the transformation endpoint is di- rectly or indirectly related to tumorigenesis per se. Thus, most transformed BALB/c (and NIH) 3T3

and C3HlOTl/2 cells are tumorigenic in nude mice (Kakunaga and Yamasaki, 1985) and some of the

morphologically transformed SHE cells can acquire tumorigenicity after subsequent passages (Barrett and Ts’o, 1978).

Again, because they were developed to simulate biological processes of carcinogenesis, cell-transfor-

mation systems are considered to respond to carcino- gens with different mechanisms of actions. In view of the increasing level of concern that ‘non-geno- toxic’ events are critically involved in carcinogenesis

and that many carcinogens induce non-genotoxic rather than genotoxic carcinogenic effects, it appears that cell-transformation systems are ideally suited for detecting both types of carcinogen (Yamasaki, 1995). However, unlike other short-term tests for carcino-

gen detection, such as the Ames test or chromosomal aberration test, the use of particular in vitro cell- transformation assays has never been sustained. Strong hopes for use of BHK cell-transformation

assays as a simple carcinogen screening method during the late 1970s (Styles, 1977) failed to materi- alize. Then, an SHE assay with cryopreserved em- bryonic cells was proposed by Pienta and his col- leagues (Pienta, 19801, and C3HlOT1/2 and BALB/c 3T3 cell-transformation systems were de-

veloped and proposed as possible carcinogen assays (Heidelberger et al., 1983). For the latter two sys- tems, international agreement for assay procedures and focus-scoring criteria have been reached (Kakunaga and Yamasaki, 1985). In spite of the biological relevance of these transformation assays

0027-5107/96/$15.00 Copyright 0 1996. Published by Elsevier Science B.V.

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and efforts of various laboratories. none of them has

entered into regular. widespread use as a screening test. The reasons for this failure of cell-transforma-

tion assays to obtain a sustained citizenship in car- cinogen screening could include: ( 1) the presence of

certain ambiguity in judging transformed cells: (21 the greater time and skill required to perform assays

than with mutation assays; and (31 the absence of convincing inter-laboratory reproducibility studies.

In this issue. LeBoeuf’s group present their at- tempts to improve the use of SHE cell transforma-

tion for detecting carcinogens. In the past, SHE cell transformation was used to screen a number of agents.

but those studies suffered from the fact that the number of transformed colonies was too low to provide credible results (Barrett et al.. 19861. LeBoeuf’s group made a major improvement to cir- cumvent this deficiency; they succeeded in establish- ing more sensitive SHE cell-transformation assays

by lowering the culture pH to 6.7 (LeBoeuf and Kerckaert. 19871. This assay allowed them to show the dose-response relationships for carcinogens, an effect which had been absent from most of the previous SHE cell-transformation data.

The data base presented here is impressive and provides a promising signal for the use of the modi- fied SHE cell-transformation protocol for the detec-

tion of carcinogens which operate through genotoxic or non-genotoxic mechanisms. Recently, a similar data base for BALB/c 3T3 cell transformation has

been provided by Matthews et al. (19931. Whether this modified SHE cell-transformation

protocol will gain wide and sustained support from the scientific and regulatory communities depends on further efforts in the laboratories involved. For ex- ample, it is important that this assay be used in other laboratories: international agreement for assay proce- dures and colony-scoring criteria should be reached.

An important aspect in establishing a reliable test for carcinogens is to have mechanistic information about the chosen assays. For example, why does a lower pH transform SHE cells more efficiently? This is partially addressed by LeBoeuf’s papers, but further studies by them, as well as by other investigators. would further strengthen the scientific basis of the new SHE cell assays.

The use of cell transformation may receive more attention in the near future in view of our changing

strategies toward carcinogen identification and risk

assessment. It has become clear that we need to incorporate more mechanistic information in addition

to results from long-term and short-term assays, in

order to undertake more reliable hazard identification

and risk assessment of human carcinogens (IARC, 1991). In this sense, we are starting to ‘study’ chemi- cals rather than simply to ‘test’ them. At this cross-

roads, cell transformation again appears to occupy a unique place, since chemicals can be studied for their involvement at different stages of transformation and

can be tested for their transforming ability.

References

Barrett. J.C. and Ts‘o. P.O.P. (1978) Evidence for the progressive

nature of neoplastic transformation in vitro. Proc. Natl. Acad.

Sci. USA, 75. 3761-3765.

Barrett, J.C.. Kakunaga, T.. Kuroki. T.. Neubett. D.. Trosko. J.E..

Vusiliev. J.M.. Williams. G.M. and Yamasaki, H. (19861

Mammalian cell transformation in culture. In: R. Montesano.

H. Bartsch. H. Vainio, J. Wilboum and H. Yamasaki (Eds.).

Long-term and short-term assays for carcinogens: a crittcal

appraisal. IARC Scientific Publications No. 83. Lyon. France,

pp. 267-786.

Berwald. Y. and Sachs. L. (1963) In vitro cell transformation with

chemical carcinogens. Nature. 200. I 182-l 184.

Goldfarb. M.. Shimizu. K., Perucho, M. and Wigler. M. (1982)

Isolation and preliminary characterisation of a human trans-

forming gene from Y24 bladder carcinoma ceils. Nature. 296.

404-40’).

Heidelberger, C.. Freeman. A.E.. Pienta, R.J., Sivak, A.. Bertram.

J.S.. Casto, B.C., Dunkel, V.C.. Francis, M.W.. Kakunaga. T.,

Little. J.B. and Schechtman. L.M. (1983) Cell transformation

hy chemical agents: a review and analysis of the literature.

Mutation Res., 114. 383-385.

Higashi, T., Sasat. H.. Suzuki, F.. Miyoahi. J. and Kakunaga. T.

t 1990) Hamster cell line suitable for transfection assay ot

transforming genes. Proc. Natl. Acad. Sci. USA. 87. 2409-

2413.

Kakunaga, T. and Yamasaki. H. (Eds.) (1985) Transformation

assay of established cell lines: mechanisms and applications.

IARC Scientific Publications No. 67. Lyon, France.

IARC (1991) Mechanisms of carcinogenesis in risk identification.

lARC Technical Report 91 /(JO?, Lyon.

LeBoeuf, R.A. and Kerckaert. G.A. (1987) Enhanced morphologt-

cal transformation of early passage Syrian hamster embryo

cells cultured in medium with a reduced bicarbonate concen-

tration and pH. Carcinogenesis, 8, 689-697.

Matthews. E.J.. Spalding. J.W. and Tennant. R.W. (1993) Trans-

formation of BALB/c 3T3 cells: V. Transformation responses

of 16X chemicals compared with mutagenicity in Salmonella

H. Yamasaki/Mutation Research 356 (1996) 1-3 3

and carcinogenicity in rodent bioassays. Environ. Health Per-

spect. 101 (Suppl 2.). 347-482.

Pienta, R.J. (1980) Transformation of Syrian hamster embryo cells

by diverse chemicals and correlation with their reported car-

cinogenic and mutagenic activities. In: F.D. de Serres (Ed.).

Chemical Mutagens, Vol. 6, Plenum, New York, pp. 175-202.

Pulciani, S.. Santos. E.. Louver, A.V.. Long, L.K.. Robbins. K.C.

and Barbacid, M. (1982) Oncogenes in human tumor cell

lines: molecular cloning of a transforming gene from human

bladder carcinoma cells. Proc. Natl. Acad. Sci. USA. 79,

2845-2849.

Shih, C. and Weinberg, R.A. (1982) Isolation of a transforming

sequence from a human bladder carcinoma cell line. Cell, 29.

161-169.

Styles. J.A. (1977) A method of detecting carcinogenic organic

chemicals using mammalian cells in culture. Br. J. Cancer, 36.

558-563.

Yamasaki, H. (1995) Non-genotoxic mechanisms of carcino-

genesis: studies of cell transformation and gap junctional

intercellular communication. Toxicol. Lett., 77, 55-61.