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Isolation of 12-O-tetradecanoylphorbol-13-acetate-resistant mutants of a macrophage-like cell line: evidence for induction by 12-O-tetradecanoylphorbol-13-acetate of a non-colony-stimulating growth factor.

There has been much recent interest in the role of a calcium-dependent, phospholipid-dependent protein kinase (PKC) in regulation of macrophage function and growth. In order to better define the role of PKC in these processes we have developed mutants of the RAW264 macrophage-like cell line that are resistant to the growth-inhibiting effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent stimulator of PKC activity. Concentrations of TPA of 10(-7) M or greater resulted in more than 90% inhibition of growth of RAW264 cells and were used to select for TPA-response mutants. Ultraviolet mutagenesis of 10(6) cells and growth selection under inhibitory TPA concentrations yielded 12 colonies. Two isolated lines (M11 and M12) were studied in detail. Neither cell line was deficient in, nor appeared to have an altered PKC, based on enzymatic activity in response to TPA or diacylglycerol. Both cell lines could grow in the presence of 10(-6) M TPA. After removal of TPA, mutant M11 continued to grow while M12 died. Growth of M12 cells was TPA concentration dependent. Flow cytometric analysis of the DNA content of M12 cells in the absence of TPA indicated that cell growth was arrested in G0 or G1. This was interpreted as indicating that TPA acted as a growth factor inducing cells to enter the cell cycle. M12 cells could also grow in L-cell-conditioned medium containing colony-stimulating factor-1 (CSF-1), the normal growth factor for cells of the macrophage lineage. After subcloning M12 cells, it was found that several subclones of M12 did not grow in response to TPA but did grow well in L-cell medium. These cells would grow in medium conditioned by the exposure of TPA-responsive subclones to TPA. Bone marrow culture cells also grew in the conditioned medium. The growth factors produced by the TPA responsive subclones were not neutralized by anti-CSF-1 antiserum. These results suggest that TPA may not directly induce the growth of M12 cells or the formation of colonies of bone marrow cells, but instead may act through the induction of a non-CSF-1 growth factor. These mutants can be used as tools for future studies on the role of PKC in the regulation of macrophage growth.