ELaboration of an optimal in vitro system to detect chemicals interfering with gap junction intercellular communication (GJIC), inhibitors or enhancers, and validation of this system (using human cells, as well as animal cells), to study mechanisms of GJIC and their role in the promotion phase of the carcinogenesis process.
Published data suggest that alteration of cell cell communication mediated through gap junctions plays an important role in carcinogenesis, especially in the tumour promotion stage. When gap junctions (cell membrane channels) are inhibited, cell growth based on the intercellular exchanges of messengers is dysregulated and can lead to an apparent tumour when exposed to a tumour promoting factor. Therefore, modulation of gap junctional intercellular communication (GJIC) will be used as the end point of a screening assay for potential tumour promoters (inhibitors of GJIC) and antipromoters (enhancers, protectors of GJIC).
In a preliminary collaboration over the in vitro GJIC assay, we were able successfully to perform a blind trial to assess the reliability of the test. 20 coded compounds, including known tumour promoters (nongenotoxic carcinogens) and non tumour promoters have been tested in 5 laboratories with the standardized microinjection dye transfer assay in vitro. The model cell system used is a rat epithelial cell line (IAR20) which has good GJIC capacity. The results of this assay evaluation demonstrate that this given test system could be suggested as one of the short term tests available for detecting tumour promoting related activity. In the area of in vitro models for early detection of nongenotoxic carcinogens, this assay is relevant to industrial use and it can easily be transferred for toxicity testing of chemicals. The protocol is standardized, reproducible and simple to perform with a large variety of cell systems (tissue specificity).
The functionality of the GJIC assay gave us a good platform to increase the sensitivity of the test in developing cell systems (HeLa transfectant cells) expressing other specific connexins (gap junctional proteins) than those expressed in IAR20 (connexin43 -cx43) and to understand individual cx sensitivity to tumour promoter action. A possible upregulation of cx expression, suggested with some compounds, could be a potentially important mechanism of cancer prevention.
Comparative results were obtained during this study. When IAR20 cells were treated with the highest non-cytotoxic concentrations of tumor promoters; 12-o-tetradecanoyl-phorbol-13-acetate(TPA); butylated hydroxyanisole(BHA); dichlorodiphenyl-trichloroethane (DDT); and phenobarbital (PB) a dose and time related inhibition of GJIC was observed. A 1 h treatment with 100ng/ml of the non promoter, 4alpha-phorbol-12,13-dicdecanoate(4alpha-PDD) caused no inhibition on GJIC.
Standardization of the dye transfer protocol is considered achieved and the assay system is functional. Studies are to be performed with liver and skin cells to explore the mechanisms of GJIC.
The impetus for the proposed project is based on the following information: 1) the detection of compounds that promote the process of carcinogenesk (tumour promoters) is an important public health objective; 2) the detectior of compounds that can act as anti-promoters has implications for chemotherapy or chemo prevention; 3) there is presently no validated in-vitro assay for the detection of promoters or anti-promoters; and 4) the disturbance of gap junctional intercellular communication GJIC has been strongly implicated as a key factor in carcinogenesis, particularly in the tumor promotion stage. Therefore, it is proposed to employ an in-vitro GJIC assay system in an extensive inter-laboratory validation trial. The work programme is focused or 1) use of the dye-coupling GJIC assay ;2) intra-laboratory testing of E limited number of pre-selected in-vivo tumor promoters and anti-promoters, using a range of human, rodent and bovine cell systems, with each laboratory using the same chemicals but different cell systems ;3) selecting from this intra-laboratory phase a test system comprising a small number of cell types to be applied to a large scale inter-laboratory blind trial of the ability of the in-vitro GJIC assay to predict promoter (inhibited GJIC), anti-Promote: (enhanced GJIC) or non-promoter (no effect) activity of a large number on chemicals of known in-vivo activity ;4) evaluation of the validity of the test systems ;5) concomitant studies of the molecular and biochemical mechanisms involved in the chemically-induced modulation Ofgjic,such studies being pivotal in understanding the role of GJIC in carcinogenesis and in designing future assays of promoter or anti-promoter activity.
Funding SchemeCSC - Cost-sharing contracts
171 77 Stockholm