DEVELOPMENT OF A SENSITIVE IN-VITRO METHOD FOR IDENTIFYING TUMOR PROMOTERS

The identification and characterization of nongenotoxic carcinogens re presents a significant challenge to toxicologists. In vitro methods fo r identifying tumor promoters with suitable sensitivity and specificit y have been particularly elusive. Experiments are described which sugg est that the human promyelocytic leukemia cell line HL-60 provides a s ensitive indicator of promoter-induced changes to gene regulation and expression. As a result of differentiation these cells undergo a trans ition from a non-phagocytic suspension culture to an attached fibrobla st-like culture which exhibits high phagocytic activity. Fluorescent l atex particles were used as sensors to highlight the phagocytic phenot ype and permitted the use of flow cytometry to automatically quantitat e particle internalization. To evaluate specificity, HL-60 cells were treated with a series of phorbol esters covering a range of in vivo tu mor promoting activity. Results indicate that this family of compounds induces HL-60 cells to differentiate in proportion to their in vivo p romoting activity. To closely assess the sensitivity of the phagocytic endpoint, HL-60 cells were treated with picogram levels of 12-O-tetra decanoyl phorbol-13-acetate (TPA), whereupon increments as low as 50 p g of TPA per mi caused statistically significant increases in phagocyt ic activity. The experiments described herein suggest that in vitro di fferentiation of HL-60 cells may reflect the promoter-dependent modifi cations to gene expression that are observed in vivo during the promot ion phase of carcinogenesis. The described method may represent a sens itive promoter screening assay which is both rapid and economical.