Purification of a serum factor which reverses dibutyryl cAMP induced differentiation

We have previously shown [Grabham et al. (1988) Expt. Eye Res. 47, 123-133] that the adenovirus 12 transformed human retinoblast cell line (Ad 12 HER 10), like a number of other cell types of neuroepithelial origin, can be induced to differentiate in response to exposure to dibutyryl cAMP, and that this differentiation can be rapidly reversed by foetal calf serum. We present data here to show that a single protein, which we have termed differentiation reversal factor (DRF) and have isolated from serum, is responsible for this activity. Following reversal by DRF the growth rate of these cells was shown to be stimulated in serum-free medium. Using ammonium sulphate fractionation, gel filtration chromatography (Ultrogel AcA44), anion exchange chromatography (DEAE cellulose) and preparative gel electrophoresis, DRF has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis in the presence and absence of SDS. The protein has a mol. wt of 72,000 and appears to exist in vivo as a monomer. The concentration of DRF in serum is in the range 100-500 micrograms/ml and is capable of reversing cAMP-induced differentiation of various primary human neuroepithelial cells at physiological concentrations.