Kiyoshi Sasaki

Cell transformation assays for prediction of carcinogenic potential: state of the science and future research needs

Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identification of potential chemical carcinogens. Despite showing good correlation with rodent bioassay data, concerns over the subjective nature of using morphological criteria for identifying transformed cells and a lack of understanding of the mechanistic basis of the assays has limited their acceptance for regulatory purposes. However, recent drivers to find alternative carcinogenicity assessment methodologies, such as the Seventh Amendment to the EU Cosmetics Directive, have fuelled renewed interest in CTAs. Research is currently ongoing to improve the objectivity of the assays, reveal the underlying molecular changes leading to transformation and explore the use of novel cell types. The UK NC3Rs held an international workshop in November 2010 to review the current state of the art in this field and provide directions for future research. This paper outlines the key points highlighted at this meeting.

A Bhas 42 cell transformation assay on 98 chemicals: The characteristics and performance for the prediction of chemical carcinogenicity

The Bhas 42 cell transformation assay is a short-term system using a clone of the BALB/c 3T3 cells transfected with an oncogenic murine ras gene (v-Ha-ras). The assay has previously been reported to be capable of detecting the tumor-initiating and tumor-promoting activities of chemical carcinogens according to the different protocols, an initiation assay and a promotion assay, respectively. We applied this short-term assay to 98 chemicals to characterize the assay and evaluate its performance for the detection of chemical carcinogenicity. When the assay results were compared with the existing genotoxicity data, the Bhas 42 cell transformation assay could detect a considerable number of Ames-negative and Ames-discordant carcinogens: and the promotion assay detected most of those Ames-negative and -discordant carcinogens. This fact suggested that the Bhas 42 cells behaved as initiated cells in the transformation assay. The performance indices were calculated from the assay results of 52 carcinogens and 37 non-carcinogens. The concordance was 78%, sensitivity 73%, specificity 84%, positive predictivity 86%, negative predictivity 69%, false negative 27% and false positive 16%. Of these values, the concordance, specificity, negative predictivity and false positive were superior and the other performance indices were equivalent to those of conventional genotoxicity tests. From overall results, we concluded that the accuracy of prediction of chemical carcinogenicity would be improved by introducing the Bhas 42 cell transformation assay into the battery of in vitro assays.

ESTABLISHMENT OF A HIGHLY REPRODUCIBLE TRANSFORMATION ASSAY OF A Ras-TRANSFECTED BALB 3T3 CLONE BY TREATMENT WITH PROMOTERS

In the detection of cancer-inducing agents, it is important to establish appropriate screening systems, not only for initiators but also for promoters. Almost all the initiators can be detected by short-term tests using bacteria, such as the Ames test, or cultured mammalian cells, such as cytogenetic tests and drug-resistant mutation tests (4). In recent years, some studies indicate that active ras genes act as initiators in two-stage carcinogenesis in vivo as well as in vitro (1, 2, 3, 7). We cloned v-Ha-ras-transfected BALB 3T3 cells (Bhas 42) by co-transfection with pSV2-neo genes. Bhas 42 cells were found to be sensitive to contact inhibition, but also to undergo a drastic transformation by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) (7). These results suggest that Bhas 42 cells are initiated cells in two-stage transformation. Therefore, we tried to apply Bhas 42 cells to screening of promoters by using the two-stage transformation assay.

The Inhibitory Effect of the Combination of Antineoplaston A-10 Injection with a Small Dose of cis-Diamminedichloroplatinum on Cell and Tumor Growth of Human Hepatocellular Carcinoma

The inhibitory effects of a combination of Antineoplaston A‐10 Injection with a small dose of m‐diamminedichloroplatinum (CDDP) on cell and tumor growth was tested in in vitro and in vivo settings. A human hepatocellular carcinoma cell line (KIM‐1) was used for the cell growth and transplanted tumor growth studies. In the cell growth study, one‐hour exposure of KIM‐1 cells to CDDP in the medium at concentrations of 0.5, 1.0, and 2.0 μg/ml inhibited cell growth dose‐dependently. Continuous exposure of cultured cells to Antineoplaston A‐10 Injection at concentrations of 4, 6, and 8 mg/ml also inhibited tumor growth dose‐dependently. The combination of 0.5 μg/ml CDDP and 6 mg/ml A‐10 Injection inhibited cell growth more than did each agent individually. Electron microscopic study showed well‐maintained organelle structures in Antineoplaston A‐10 Injection‐treated cells compared to CDDP‐treated cells. α‐Fetoprotein (AFP) production by 104 cells in 48 h increased in the A‐10 Injection‐treated and A‐10 Injection + CDDP‐treated groups as the concentration of these agents increased. In the tumor growth study, daily administration of Antineoplaston A‐10 Injection 75 mg with once a week administration of 20 μg of CDDP for 5 weeks inhibited transplanted tumor growth in athymic mice after 33 days of treatment, while administration of 75 mg of A‐10 Injection or 20 or 60 μg of CDDP alone showed no significant inhibition of tumor growth.

Comparison of sensitivity to arsenic compounds between a Bhas 42 cell transformation assay and a BALB/c 3T3 cell transformation assay.

A short-term cell transformation assay has recently been developed, using Bhas 42 cells which were established from BALB/c 3T3 cells transfected by v-Ha-ras gene and postulated to be initiated in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay has been reported to be capable of detecting initiating and promoting activities of chemical carcinogens, according to the different protocols, initiation assay and promotion assay, respectively. The assay is superior to classical transformation assays in cost and labor performance. The present study was carried out to compare its sensitivity with that of a classical BALB/c 3T3 cell system. We performed the Bhas 42 cell transformation assay with inorganic arsenic compounds which are potent environmental carcinogens in human but not mutagens in bacteria or weak mutagens in mammalian cells in vitro. Sodium arsenite, disodium arsenate, and their metabolites, monomethylarsonic acid and dimethylarsinic acid (DMAA) were included in the study. Sodium arsenite was positive in the initiation assay and all compounds except for DMAA were positive in the promotion assay. These results were compared with reported data in a two-stage BALB/c 3T3 cell transformation assay. The sensitivity of Bhas 42 cell transformation assay was found to be similar to that of the conventional BALB/c 3T3 cell transformation assay for the detection of initiating activities of arsenic compounds. For the detection of promoting activities, its sensitivity was equivalent to that of the two-stage BALB/c 3T3 cell transformation assay where the target cells were initiated with sub-threshold dose of 3-methylcholanthrene, confirming that Bhas 42 cells behave as initiated cells in the transformation assay.

Demonstration by DNA fingerprint analysis of genomic instability in mouse BALB 3T3 cells during cell transformation

We employed DNA fingerprint analysis to monitor DNA rearrangements in BALB 3T3 cells transformed spontaneously or by treatment with 3-methylcholanthrene (MCA) and UV-C. The effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in combination with MCA was also examined. Twenty-three spontaneously transformed cells, 28 induced transformed cells (18 by 1 microgram/ml MCA, six by 5 micrograms/ml MCA, and four by UV-C), and 31 non-transformed subclones were isolated from parental BALB 3T3 A31-1-1 cells. The DNAs were digested with HinfI and subjected to DNA fingerprint analysis with three multi-locus minisatellite probes, Per-6, Core, and Ins. Per-6 was the most effective probe for detecting DNA rearrangements. Rearranged bands detected by the Per-6 probe were observed in 9/31 (29%) of non-transformed subclones, 14/23 (61%) of spontaneously transformed cells, 16/18 (89%) of cells transformed by 1 microgram/ml of MCA, 6/6 (100%) of cells transformed by 5 micrograms/ml MCA, and 4/4 (100%) of UV-C-transformed cells. Higher numbers of DNA rearrangements (> or = 3) occurred most frequently in the induced transformed cells. TPA enhanced the frequency of DNA rearrangements in cells transformed by MCA. These data indicate that (1) genomic DNA in BALB 3T3 cells is unstable and susceptible to rearrangement, (2) its instability is elevated during cell transformation, and (3) MCA and UV-C induce DNA rearrangements, and TPA enhances the effect of the former, probably via the recombination process. DNA fingerprint analysis is valuable for monitoring genomic instability during cell transformation.

Regulation of G418 Selection Efficiency by Cell-Cell Interaction in Transfection

We attempted to establish the optimum conditions for the calcium phosphate (CaPO4) precipitation protocol by counting G418 resistant (G418r) colonies after transfection of pSV2-neo DNA into BALB 3T3 cells. The amount and molecular size of carrier DNA, number of plating cells, treatment period of DNA-CaPO4 precipitates and expression time of G418 selection were found to be important factors in the induction of G418r colonies. Six G418r clones were derived from BALB 3T3, NIH 3T3 and FRSK cells, and cocultured with G418 sensitive (G418s) parent cells in G418 medium. The colony formation capacity of all G418r cell clones decreased with the increasing number of plated G418s cells. Cell-cell contact appeared to be necessary to reduce the colony formation of G418r cells, and contact-dependent G418r cell killing was probably not related to gap junction formation. Contact-mediated cell killing is a likely explanation for the observation that induction of G418r colonies is often reduced under conditions of high-density plating, long treatment of DNA-CaPO4 precipitates, and long expression time of G418 selection. These results suggest that in some instances transfection efficiency using pSV2-neo DNA should be carefully evaluated because culture conditions can mask the induction of G418r colonies.

GENE TRANSFER INTO MAMMALIAN CELLS BY RAPID FREEZING

SummaryIn this paper, we describe a simple technique to introduce DNA into cells through cracks and/or pores in cell membranes caused by intracellular ice crystal formation induced by liquid nitrogen. We mixed mouse BALB 3T3 cells and pSV2-neo DNA and froze the cell suspension under various conditions to determine those optimum for the introduction of DNA into mammalian cells. We found that brief treatment with liquid nitrogen, which showed only moderate cell killing, resulted in the induction of G-418 resistant colonies. These results suggest that this new technique is useful for transfection of genes into mammalian cells.