MITOGENIC EFFECTS AND NUCLEAR-LOCALIZATION OF PROCORTICOTROPHIN-RELEASING HORMONE EXPRESSED WITHIN STABLY TRANSFECTED FIBROBLAST CELLS (CHO-K1)

To investigate the intracellular localisation and biological activity of procorticotrophin-releasing hormone (proCRH), we have established s tably transfected CHO-K1 cells expressing the rat pre-proCRH cDNA. Usi ng immunoblot analysis of cell lysates of transfected CHO-K1 cells, we detected a major CRH immunoreactive band with an apparent molecular w eight of approximately 19 kDa. This 19 kDa band could account for full length proCRH molecule which has not undergone post-translational mod ifications. Metabolic labelling followed by immunoprecipitation, SDS-P AGE and autoradiography indicated that no endoproteolytic processing o f proCRH takes place within the transfected CHO-K1 cells. Immunofluore scence staining localises the CRH precursor to both the cytoplasm and to the nucleus in transfected CHO-K1 cells. This result was confirmed using subcellular fractionation techniques on radiolabelled CHO-K1 cel ls expressing immunoreactive CRH. A major CRH-immunoreactive band of 1 9 kDa was detected both in the microsomal and secreted fractions, indi cating the presence of proCRH within the secretory pathway of these ce lls. This was also evident in the nuclear fraction, therefore confirmi ng the nuclear localisation of proCRH. Analysis of DNA concentration, cell number and DNA synthesis showed that stably transfected CHO-K1 ce lls expressing proCRH have a higher proliferation and DNA synthesis ra te than wildtype CHO-K1 cells or CHO-K1 cells transfected with pEE14 a lone. Our results therefore suggest a mitogenic role for the intact pr oCRH molecule within CHO-K1 cells. Furthermore, treatment of mouse cor ticotrophic tumour cells (AtT20/D16-16) with conditioned medium from t ransfected CHO-K1 cells expressing proCRH, stimulated both DNA synthes is and cell proliferation above basal levels. Our results constitute t he first reported direct evidence of a mitogenic role for proCRH actin g on a corticotrophic cell population.