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Characterization of a human teratocarcinoma cell assay for inhibitors of metabolic cooperation
Abstract:
Although a Chinese hamster V79 cell-based assay for inhibitors of metabolic cooperation is currently available, the development of a human cell-based assay is desirable in order to avoid inappropriate extrapolation from animal cells to human cells. Cells derived from a human teratocarcinoma cell line (designated PA-1), which has a stable pseudodiploid karyotype and excellent in vitro growth properties, were used in a metabolic cooperation assay. The assay was based on the metabolic isolation of hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient variants in the presence of HGPRT-proficient cells and 6-thioguanine. Chemicals which inhibit the transfer of the lethal metabolite of 6-thioguanine from HGPRT-proficient to HGPRT-deficient cells will allow for recovery of the 6-thioguanine-resistant (HGPRT-deficient) cells. Chemicals tested included 12-O-tetradecanoylphorbol-13-acetate and related analogues phorbol-12,13-didecanoate, mezerein, and 4-phorbol-12,13-didecanoate. Concurring with results previously obtained in V79 cells, 12-O-tetradecanoylphorbol-13-acetate and phorbol-12,13-didecanoate strongly inhibited metabolic cooperation, whereas mezerein was moderately inhibitory and 4 alpha-phorbol-12,13-didecanoate was inactive. These cells thus hold promise as a human cell-based assay for inhibitors of metabolic cooperation.
Insights
A new human cell assay using PA-1 cells was developed to study metabolic cooperation. This assay effectively identifies inhibitors of metabolic cooperation, similar to existing animal cell assays.
Area of Science:
- Cell Biology
- Toxicology
- Biochemistry
Background:
- A validated Chinese hamster V79 cell assay exists for metabolic cooperation inhibitors.
- A human cell-based assay is needed to avoid extrapolation issues from animal to human cells.
Purpose of the Study:
- To develop and validate a human cell-based assay for inhibitors of metabolic cooperation.
- To assess the utility of the PA-1 human teratocarcinoma cell line in this assay.
Main Methods:
- Utilized the PA-1 human cell line in a metabolic cooperation assay.
- The assay involved metabolic isolation of hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient cells using 6-thioguanine.
- Tested phorbol ester analogues, including 12-O-tetradecanoylphorbol-13-acetate, phorbol-12,13-didecanoate, mezerein, and 4-phorbol-12,13-didecanoate.
Main Results:
- 12-O-tetradecanoylphorbol-13-acetate and phorbol-12,13-didecanoate strongly inhibited metabolic cooperation.
- Mezerein showed moderate inhibition, while 4-phorbol-12,13-didecanoate was inactive.
- Results aligned with previous findings in V79 cells.
Conclusions:
- The PA-1 cell line is a promising candidate for a human cell-based assay for metabolic cooperation inhibitors.
- This human cell assay can provide more relevant data for human health risk assessment.