Placental keratinocyte growth factor: partial purification and comparison with epidermal growth factor.
A water-soluble extract of term human placenta, which was previously shown to promote proliferative growth of human keratinocytes in defined medium, enhanced both cellular attachment and proliferative growth. We have partially purified the activity which enhanced cell growth and examined its action in keratinocytes. Activity was precipitated from the crude extract by (NH4)2SO4 between 33 and 60% saturation and chromatographed by gel filtration. The activity did not bind to heparin-Sepharose at low ionic strength but was adsorbed to DEAE-cellulose from which it was eluted with NaCl and then passed over phenyl-HPLC to remove bovine serum albumin previously added to protect the activity. The active fraction was applied to gel exclusion HPLC in the presence of 0.02% octyl-beta-D-glucopyranoside, which yielded an apparent Mr 35,000 for the factor. Purification was approximately 200-fold with approximately 4% recovery. The factor appears to be a protein, since activity is destroyed by trypsin. Autoradiography of cultures treated with the placental factor or epidermal growth factor (EGF) revealed that approximately 50% of cells were labeled after treatment with either growth factor compared to 9% in control cultures after a [3H]thymidine pulse. Protein synthesis was increased by about 50% 42 h after treatment with either agent, consistent with a 50% increase in nuclear labeling. Cell number was increased fivefold after 6 days in the presence of the partially purified factor, whereas EGF increased cell number eightfold. Stimulation of [3H]thymidine incorporation by the partially purified factor, in contrast, was about twice that produced by EGF, indicating that thymidine incorporation is preferentially stimulated by the placental factor and does not correlate well with other parameters of proliferative growth. The placental keratinocyte growth factor is a unique factor with a novel effect on incorporation of thymidine into DNA.
Department of Dermatology, University of North Carolina, School of Medicine, Chapel Hill 27514.
M L
ML
Chiu
E J
EJ
O'Keefe
D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004817:Epidermis / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008715:Methionine / Q000378:metabolism; D011257:Pregnancy Proteins / Q000302:isolation & purification*; D013936:Thymidine / Q000378:metabolism
D011257:Pregnancy Proteins; D004815:Epidermal Growth Factor; D008715:Methionine; D013936:Thymidine