Differentiation-inducing factor

Abstract: To understand pattern formation in an organism one needs to know what factors control the differentiation of each cell type and what elements are responsible for the spatial arrangement of these cell types. In the cellular slime mould Dictyostelium discoideum, the problem is relatively simple since the mature fruiting body consists of two basic cell types—stalk cells and spores. The stalk cells of the mature fruiting body are derived from the anterior cells of the multicellular masses formed by aggregation, whereas the spores are derived from the posterior cells1. Cells that are plated at a density too low for aggregation, or are allowed to aggregate under water do not normally differentiate into stalk or spore cells. In many of our experiments we have made use of the fact that cells plated on agar at any density fail to undergo development beyond the stage of aggregation if they are covered by a thin layer of Cellophane. In this paper we present observations on cell differentiation which derive from the original report of Bonner2 of stalk-cell induction by cyclic AMP in isolated cells. We have found that stalk-cell induction by cyclic AMP is markedly dependent on cell density, and present evidence for the involvement of a low molecular weight diffusible factor in this process. We also describe the isolation of a mutant which gives rise to spore cells under cellophane.

Abstract: During development in the cellular slime mould Dictyostelium discoideum starved amoebae aggregate to form multicellular structures that display a simple antero-posterior pattern: pre-stalk cells occupy the front 20% of the aggregate, and prespore cells occupy the remainder1–4. We have attempted to elucidate the nature of the mechanism regulating the proportions of the two cell types1,5,6 by examining the factors that influence the pathway of differentiation of amoebae in vitro. Amoebae of D. discoideum strain V12 M2 form stalk cells efficiently in appropriate conditions and ‘sporogenous’ derivatives produce spores as well as stalk cells7,8. Mature spores are formed in a medium containing only cyclic AMP and salts9, whereas formation of stalk cells requires, in addition, a low molecular weight hydrophobic factor (DIF)7,10. Recent observations have led us to propose that DIF is a morphogen responsible for activating stalk cell differentiation5,11–13. Here we present evidence that ammonia is a second morphogen, that acts antagonistically to DIF, and that the choice of differentiation pathway is mediated by intracellular pH.

Abstract: Human myelogenous leukaemic cells can be induced to differentiate into the monocyte/macrophage pathway by protein inducers called differentiation inducing factors (DIF) in conditioned media of mitogen–stimulated human peripheral blood leukocytes1–8. However, human DIF has not yet been well characterized. DIF is known to be a T-cell lymphokine, as it can be obtained from the T-cell line HUT-1027 and can be partially purified from medium conditioned by phytohaemagglutinin (PHA)–stimulated lymphocytes8. We found that monocytes also produce factor(s) that induce differentiation of human myelogenous leukaemia cell lines to cells with macrophage-like characteristics. This factor(s) has activity different from that of colony–stimulating factor(s) or interferons9 . We have now purified a DIF to homogeneity from medium conditioned by PHA–stimulated leukocytes using a human myeloblastic leukemia cell line, ML-110, as target cells. The purified DIF has a relative molecular mass (Mr) of approximately 17,000, with an NH2-terminal sequence the same as that of human tumour necrosis factor (TNF). Recombinant human TNF (rHuTNF) induces differentiation of ML-1 cells and an anti-pDIF monoclonal antibody can neutralize both differentiation inducing activity and cytotoxic activity of DIF and rHuTNF. The findings indicate that one of the DIF(s) produced by leukocytes is probably TNF.

Abstract: Spleen cells treated with mitogens produce a potent bone-resorbing factor called osteoclast-activating factor (OAF). To examine the relationship between the bone-resorbing factor and other protein factors produced by spleen cells, the colony-stimulating factor (CSF), the differentiation-inducing factor (DIF), the macrophage fusion factor (MFF), and the macrophage growth factor (MGF) were purified from 2.68 liters of conditioned medium of mouse spleen cell cultures treated with concanavalin A. Purification was performed successively by DEAE-cellulose, Blue Sepharose, and Sephadex G-150 column chromatography and high-pressure liquid chromatography (HPLC). The DIF was successfully separated from CSF and MGF on HPLC. CSF coincided with MGF on HPLC, but MFF disappeared before application to HPLC. Only the DIF exhibited bone-resorbing activity, whereas CSF and MGF did not. The DIFs purified from L929 cells and Ehrlich ascites tumors similarly exhibited bone-resorbing activity. The DIFs purified from spleen cells and Ehrlich ascites tumor cells exhibited neither interleukin 1 (IL-1) activity nor tumor necrosis factor (TNF) activity, though the unfractionated conditioned medium from spleen cells did exhibit them. In the light of recent reports that IL-1 beta and TNF also stimulate bone resorption, the term OAF should refer to a generic activity rather than a single factor.