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.     

Effect of placental factors on growth and function of the human fetal adrenal in vitro

Conditioned medium from human placental monolayer cultures (PM) had a marked stimulatory effect on proliferation (3H-thymidine uptake) of human fetal zone adrenal cells in primary monolayer culture, even in the absence of serum. Epidermal growth factor (EGF) and fibroblast growth factor (FGF) also significantly stimulated fetal adrenal cell growth. However, the effects of PM differed from those of EGF and FGF in several respects: 1) maximal response to PM was 2-5 times greater; 2) mitogenic effects of EGF and FGF were suppressed by adrenocorticotropic hormone (ACTH), whereas that of 50% PM was not; 3) PM inhibited ACTH-stimulated steroidogenesis (dehydroepiandrosterone sulfate and cortisol), but EGF and FGF did not. Preliminary characterization studies have indicated that approximately half of the placental growth-promoting activity is heat resistant and sensitive to bacterial proteases, and that 50-60% of the activity is lost after dialysis with membranes having a molecular weight cutoff of 3500. These findings suggest a role for the placenta in the growth and differentiated function of the human fetal adrenal gland.     

An androgen-dependent subclone derived from a mouse mammary tumor, Shionogi carcinoma 115, secretes a heparin-binding growth factor having an apparent molecular weight of 31,000 in response to androgen.

An androgen-dependent cell line denoted SC2G is a clone of an androgen-dependent mouse mammary tumor, Shionogi Carcinoma 115. Fibroblast growth factors (FGFs), epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) are stimulatory for the growth of SC2G cells in the absence of androgen. This clone was found to secrete an androgen-induced growth factor mostly eluting at 1.8 M NaCl on a heparin-Sepharose column. This factor was partially purified by chromatography on two consecutive heparin-Sepharose columns followed by cation-exchanging chromatography on an S-Sepharose column from the chemically defined serum-free medium conditioned by SC2G cells in the presence of androgen. The factor was a heat- and acid-labile cationic protein that was inactivated by reduction with dithiothreitol. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, most of the growth-promoting activity of this factor was found at approx. 31 kDa under non-reduced conditions. Neither neutralizing antibody against basic-FGF nor that against EGF inhibited the growth-promoting activity of this factor in cell culture, suggesting the factor was distinct from basic FGF or EGF. However, the possibility that the factor was another FGF- or EGF-like growth factor was not excluded.     

Are factors originating from serum,plasma, or cultured cells involved in the growth-promoting effect of the extracellular matrix produced by cultured bovine corneal endothelial cells?

The possibilities that the growth-promoting effect of the extracellular matrix (ECM) produced by cultured bovine corneal endothelial (BCE) cells could be due to: (1) adsorbed cellular factors released during the cell lysis process leading to the denudation of the ECM; (2) adsorbed serum or plasma factors: or (3) adsorbed exogenous growth factors have been examined. Exposure of confluent BCE cultures to 2 M urea in medium supplemented with 0.5% calf serum denudes the ECM without cell lysis. The ECM prepared by this procedure supports cell growth just as well as ECM prepared by denudation involving cell lysis. Thus, it is unlikely that the growth-promoting properties of ECM are due to adsorbed cellular factors. When the ECM produced by BCE cells grown in defined medium supplemented with high-density lipoprotein, transferrin, and insulin was compared to the ECMs produced by cells grown in the presence of serum- or plasma-supplemented medium, all were found to be equally potent in stimulating cell growth. It is therefore unlikely that the growth-promoting ability of the ECM is due to adsorbed plasma or serum components. When fibroblast growth factor (FGF)-coated and ECM-coated plastic dishes were submitted to a heat treatment (70 degrees C, 30 min) which results in the inactivation of FGF, the growth-supporting ability of FGF-coated dishes was lost, while the comparable ability of ECM-coated dishes was not affected significantly. This observation tends to demonstrate that the active factor present in the ECM is not FGF. Nor is it platelet-derived growth factor (PDGF), since treatment known to destroy the activity of PDGF, such as exposure to dithiothreitol (0.1 M, 30 min, 22 degrees C) or to beta-mercaptoethanol (10%) in the presence or absence of 6 M urea for 30 min at 22 degrees C, does not affect the growth-promoting activity of ECM. It is therefore unlikely that the growth-promoting effect of ECM is due to cellular growth-promoting agents or to plasma or serum factors adsorbed onto the ECM.     

Opposite effects of EGF on involucrin accumulation of A431 keratinocytes and a variant which is not growth-arrested by EGF

Summary The A431 cell line is composed of malignant keratinocytes derived from a vulval epidermo?d carcinoma. These cells have the peculiarity to stop their proliferation when they are treated with physiological concentrations of EGF, which is a mitogen for normal keratinocytes. We reported earlier that EGF induces involucrin accumulation in A431 cells and proposed that the arrest of proliferation triggers differentiation as shown by the induction of this cornified envelope precursor protein. To test this hypothesis, we compared the A431 subclone 15, which is not growth arrested by EGF-treatment, to the parental A431 cells. We found indeed that EGF reduces the involucrin content of clone 15 cells in a dose dependent manner. These opposite effects of EGF on the expression of terminal differentiation marker involucrin in A431 and A431 clone 15 keratinocytes were observed in defined medium as well as in presence of fetal calf serum. Nevertheless, when growth of parental A431 cells was inhibited by treatment with TGF-β or simply when cultures reached confluency, no involucrin accumulation was observed. Therefore growth arrestper se is not directly correlated with the induction of differentiation. Editor's Statement These results in a well-defined model system support the accepted idea that growth arrest is associated with the processes of cell differentiation, but also indicate that growth arrest alone will not lead to differentiation.     

Cell kinetic characterization of the epidermal growth factor dependent BALB/MK line using flow cytometric analysis of DNA content and iododeoxyuridine incorporation

Epidermal hyperproliferation (psoriasis, wound repair) is the result of quiescent (G₀) keratinocytes being recruited into the cell cycle. A detailed characterization of the cell cycle kinetic parameters of the mouse keratinocyte line (Balb/MK) has been carried out with the aid of bivariate iododeoxyuridine (IdUrd) and DNA analysis using flow cytometry, in order to establish whether it might provide a useful model for the study of the biochemical events controlling recruitment into the cell cycle. Balb/MK keratinocytes were cultured using low Ca²⁺ Dulbecco's modified Eagle's medium/F12 in the presence of 10% dialysed fetal bovine serum and 4 ng/ml epidermal growth factor (EGF). IdUrd labelling followed by flow cytometric analysis of trypsinized cells showed that about 95% of the population were actively cycling, with a cell cycle time of around 14 h and no significant contact inhibition. Omission of serum and EGF followed by IdUrd pulse-labelling indicated that the cells progressively withdrew from the cycle and, after 16h, less than 10% incorporated IdUrd. Subsequent restimulation with serum resulted in a synchronized cohort of cells being recruited. Entry into the S phase of the cell cycle (IdUrd incorporation) started at 8 h and was maximal between 12 h and 16 h after stimulation. Restimulation with EGF alone reached a growth fraction of 87% after 24 h continuous labelling compared with 97% using serum together with EGF. Epidermal growth factor already showed a significant stimulation at 10 pg/ml compared with the controls. There is a broad plateau centred on 5 ng/ml, followed by a slight decline above this level. We conclude that the use of a cell line with defined cell cycle kinetic parameters which can be switched between the quiescent and cycling states in a fully defined medium will provide an ideal model for biochemical studies of the relevant signal transduction pathways in keratinocytes.     

Tissue culture media; the essential non-dialyzable factors in human placental cord serum

1. Human placental cord serum contains a factor or factors necessary for active growth of fibroblasts in tissue culture and a factor which prevents the appearance of a characteristic toxicity. 2. The growth-promoting factor(s) is stable to heating to 60°C. for 30 minutes and is associated with the euglobulin fraction of the serum. 3. Extraction of dried cord serum with benzene, chloroform, and ether does not cause any decrease in growth-promoting activity. 4. Sera from hypophysectomized, adrenalectomized, and thyroid-parathyroidectomized rats show as much growth-promoting activity as normal rat sera. 5. Bovine gamma globulins, anterior pituitary growth hormone, and insulin do not replace the serum growth factor. 6. The toxicity-preventing factor is serum albumin. 7. Evidence is presented that the toxicity is due to free fatty acids in the medium and that serum albumin acts by binding the fatty acids.     

Anchorage-independent growth of normal human mesothelial cells: A sensitive bioassay for EGF which discloses the absence of this factor in fetal calf serum

Summary This laboratory recently reported that normal human mesothelial cells require epidermal growth factor (EGF) and hydrocortisone (HC), in addition to fetal calf serum and a complex defined medium component, in order to grow optimally in surface culture (9). We report here that this normal cell type also forms large colonies at high efficiency in semi-solid medium, but exhihits more stringent serum and EGF requirements for anchorage-independent than for surface growth. Mesothelial cells are unable to divide at all in semi-solid medium with added EGF or with less than 2% serum, whereas they grow slowly but progressively in surface culture under such conditions. In semi-solid medium containing 20% serum and HC, mesothelial cells are stimulated to divide by the addition of as little as 30 pg/ml purified EGF. Human urine or male mouse plasma could substitute for purified EGF, yielding growth commensurate with the levels of EGF in these biological fluids previously measured by others using radioreceptor and radioimmune assays. Thus growth of mesothelial cells in semi-solid medium can serve as a highly sensitive assay of EGF biological activity which is unaffected by the presence of serum proteins. In addition, our results demonstrate that fetal calf serum does not provide mitogenic levels of EGF to cultured cells, raising the question of the identity of plasma and serum mitogens. This work was supported by NIH grants RO1 AG02048 and RO1 CA26656 to James G. Rheinwald and by NIH postdoctoral fellowship F32 AG05303 to Paul J. La Rocca.     

Is there a ubiquitous growth factor in the eye? Proliferation induced in different cell types by eye-derived growth factors

In a previous work [1] we showed that a neutral extract of bovine adult retina RE can stimulate the growth and modify the morphology of bovine epithelial lens (BEL) cells in vitro. We were also able to demonstrate that the differences in cell shape are closely related to the cell growth properties induced by RE and are mediated by cytoskeletal protein organization as well as external proteins. In this study, we report the results of further investigations on this retinal extract. We show that it possesses all the characteristics of other growth factors such as promoting proliferation in low serum concentration or of enhancing the colony-forming efficiency of BEL cells considerably. By comparing the morphological response of BEL cells treated with RE with the response of other cells to other growth factors, we propose that the phenotypic modifications are cell specific, but not growth factor specific. We report also that RE has a broad spectrum of activity since it is able to stimulate cells from different origins and species (vascular and corneal endothelial cells, myoblasts, chondrocytes, neuroblastoma cells, and keratinocytes), but not all of them, since it can be toxic for fibroblasts. In this respect, it has an activity similar in many aspects to FGF and EGF, while it differs from them for some target cells. Its action has also been compared with the effects of retinoic acid derivatives and shown to be strikingly different. RE-like activity can be found in other ocular tissues from bovine and other species. The highest growth-promoting capacities were found in extracts of iris, pigmented epithelium with choroid, and vitreous body. The nature of all these extracts has not yet been determined. Since they are prepared in a similar way and since they have similar growth-promoting activity, we postulate that there is an ubiquitous growth factor in the eye called eye-derived growth factor (EDGF) which may play an important role in physiology and pathology of the eye.     

Proliferation of guinea-pig uterine epithelial cells in serum-free culture conditions: effect of 17 beta-estradiol, epidermal growth factor and insulin

The effects of 17 beta-estradiol (E2), epidermal growth factor (EGF) and insulin, alone or in association on guinea-pig uterine epithelial cell proliferation were examined in serum-free culture conditions. Primary cultures of epithelial cells were made quiescent by serum depletion, then incubated in a chemically defined medium. In this medium, insulin increased DNA synthesis but not in a dose-dependent manner for concentrations ranging from 0.2 to 10 micrograms/ml. A significant effect of EGF was found only for the highest concentration tested (100 ng/ml). E2 alone or in the presence of insulin (1 microgram/ml) had no effect whatsoever on the concentration tested (10(-10)-10(-5)M). Insulin (10 micrograms/ml) plus EGF (100 ng/ml) exerted on DNA synthesis and cell proliferation a significant additive effect which was identical to the growth stimulation induced by 10% fetal calf serum. The effects of insulin plus EGF were not modified by the addition of E2. These findings suggest that E2 is not directly mitogenic for uterine epithelial cells in defined culture conditions and that the mitogenic response to optimal concentration of insulin plus EGF is independent of E2.     

Human A673 cells secrete high molecular weight EGF-receptor binding growth factors that appear to be immunologically unrelated to EGF or TGF-alpha

Extracts of serum-free conditioned medium from human rhabdomyosarcoma A673 cells contain high molecular weight (HMW) transforming growth factors (TGFs) that can be partially purified by Bio-Gel P-100 and carboxymethyl (CM)-cellulose chromatography (Todaro et al: Proc Natl Acad Sci USA 77:5258, 1980). Reverse-phase high performance liquid chromatography (HPLC) revealed a principal peak of epidermal growth factor (EGF) radioreceptor assay (RRA) activity and anchorage-independent growth (AIG) activity that coeluted with 25-26% acetonitrile. If a trailing shoulder of EGF RRA activity from the CM-C chromatography was included in the material for HPLC analysis, additional active fractions were observed at 21-22% acetonitrile. Importantly, both active regions from HPLC failed to compete in radioimmunoassays under reduced and denatured conditions for human EGF (hEGF), human TGF-alpha (hTGF-alpha), or rat TGF-alpha (rTGF-alpha) and failed to give positive signals in Western blots under conditions in which TGF-alpha was readily detected when using an antisera raised against the 17 C-terminal amino acids of rTGF-alpha. Nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed EGF RRA and AIG activities in gel slices corresponding to Mr 15,000 and 22,000 in the 25-26% acetonitrile eluate and Mr 15,000, 20,000, 27,000, and 48,000 in the 21-22% acetonitrile eluate. The presence of multiple forms of EGF-receptor-binding peptides produced in vitro suggest size heterogeneity and possible immunologic diversity among high molecular weight members of the EGF/TGF-alpha family of growth-promoting polypeptides.     

Ligand- and kinase activity-independent cell survival mediated by the epidermal growth factor receptor expressed in 32D cells

To investigate the intrinsic activities of the epidermal growth factor receptor and the role of its kinase domain in these functions within a cellular environment lacking endogenous ErbB protein expression, wild-type EGF receptor (WT-EGFR) and two kinase-impaired mutants, D813A and K721R, were expressed in 32D murine hematopoietic cells, a line which is normally dependent on interleukin 3 (IL3) for growth and survival. Addition of EGF in the absence of IL3 stimulates receptor autophosphorylation and, in the presence of serum, mitosis in cells expressing WT-EGFR, but not in cells expressing D813A or K721R. Unexpectedly, cells expressing WT-EGFR or K721R exhibited IL3-independent survival in the presence of fetal bovine serum; parental 32D cells and cells expressing D813A did not survive, apparently undergoing apoptosis in the absence of IL3, whether or not serum was present. Addition of EGF did not prevent the apoptosis of WT-EGFR or K721R cells in serum-free medium. Activation of Akt was not necessary to mediate the prosurvival activity of EGF receptor expression. These results suggest that the EGF receptor can mediate the prevention of apoptosis independently of both receptor-ligand binding and receptor kinase activity, and this activity is disrupted by the D813A mutation.     

Influence of epidermal growth factor (EGF) on ruffling activity, pinocytosis and proliferation of cultivated human glia cells

Normal human glia cells in culture were studied with respect to ruffling activity, macro-pinocytosis and cell proliferation under standard culture conditions with 10% serum in the medium, in serum-free medium and after addition of epidermal growth factor (EGF) or serum to previously serum-free medium. Pinocytotic uptake of droplets of medium occurred only in relation to well developed ruffling membranes. Omitting the serum from the medium led to a drastic reduction in thymidine incorporation. The cells became slender under these conditions, and soon after the change of medium their ruffling activity and pinocytosis were almost completely abolished. Following the change to a medium containing 2 ng EGF/ml a rapid reappearance of ruffling and pinocytosis was observed. DNA synthesis, however, was not demonstrated until after 20 h, showing that ruffling and pinocytosis occurred before DNA synthesis had started. Thus EGF may initially induce conformational changes of the plasma membrane, resulting in its internalization due to formation of endocytotic vacuoles. The observed relationship between occurrence of well developed ruffling membranes, macro-pinocytosis and cell multiplication indicates that one of the functions of growth-promoting factors may be stimulation of plasma membrane turnover.     

Stimulative effect of serum on the growth of HeLa cells suppressed in a K+-deficient chemically defined medium

Growth of HeLa cells cultured with a chemically defined medium was slightly stimulated in the presence of 5% dialyzed calf serum. The growth-promoting action of serum was more conspicuous when cell growth was suppressed in the same medium, in which K⁺ was replaced by Rb⁺ to various ratios. The growth-promoting factors(s) of serum was heat-labile. Upon addition of dialyzed serum, passive K⁺ or Rb⁺ influx was increased, whereas the active cation uptake was unaffected and cell K⁺ was rather decreased. The serum did not alter uptake of [³H] amino acids. Also, protein synthesis inhibited in the Rb⁺-substituted medium was not stimulated significantly, except that observed only when the external K⁺/Rb⁺ ratio was $\text{1}\text{5}$. From the distinct effects of serum on cell growth and protein synthesis, we conclude that (i) the serum-induced stimulation of cell growth, which is suppressed in the Rb⁺-substituted medium, is not a result of the direct effect of serum on synthesis of bulk protein, but a reflection of the effect on another mechanism(s) required for cell growth; and that (ii) this action is basically identical with the growth-promoting action on cells cultured in the normal medium.     

Growth factor interactions between mouse mammary cell lines cocultured in collagen gels

Summary Three related mouse mammary cell lines were cultured in collagen gels and assayed for growth factor responsiveness and interaction via soluble factors. The CL-S1 cell line is nontumorigenic and grows poorly in collagen gel culture. The +SA and −SA cell lines exhibit different degrees of malignant behavior in vivo and have different growth properties in vitro. In collagen gel culture, +SA growth was stimulated by serum but not by epidermal growth factor (EGF), whereas both serum and EGF were required for optimal growth of −SA cells of early passage number as well as CL-S1 cells. −SA cells of later passage repeatedly exhibited a change so as to no longer require serum while retaining EGF responsiveness. [¹²⁵I]EGF binding analyses indicated that CL-S1 cells bound EGF with less affinity than did −SA cells whereas +SA cells bound almost to ligand. When cell lines were maintained in separate collagen gels but shared the same culture medium, growth of +SA or −SA cells was slightly enhanced in the presence of CL-S1 cells and −SA cell growth was enhanced by the presence of +SA cells. Using the normal rat kidney fibroblast line NRK (clone 49F) as an indicator, serum-containing conditioned media from each cell line and from each pair of cell lines cultured in collagen gels were tested for transforming growth factor (TGF) activity. Both the −SA and CL-S1 lines tested positive for TGF-α production and possibly released a TGF-β activity. These results suggest mechanisms by which cell populations in and around tumors can modify one another’s growth characteristics. The work was supported by a grant from the American Institute for Cancer Research, by American Cancer Society Institutional grant IN-119, by funds from the Poncin Trust (Seattle-First National Bank), and by grants CA-39611 and CA46885 from the National Institutes of Health, Bethesda, MD.     

Stimulation of rat placental cell DNA synthesis by transferrin

The purpose of the present investigation was to evaluate the in vitro requirements for rat placental cell DNA synthesis. A cell line established from the labyrinth region of midgestation rat chorioallantoic placentas was used to examine the actions of various agents. Transferrin was found to stimulate rat placental cell DNA synthesis and cell proliferation. The effects of transferrin on rat placental cell growth paralleled those observed with fetal bovine serum. Rat placental cells were responsive to both rat and human transferrin. Iron-saturated (holo-) transferrin was a more potent stimulator of rat placental cell DNA synthesis than was iron-free (apo-) transferrin. Addition of insulin, epidermal growth factor, or insulin-like growth factor-II to serum-free medium supplemented with rat transferrin did not significantly enhance rat placental cell DNA synthesis beyond that observed with only transferrin. The results demonstrate that a population of cells exists within the rat chorioallantoic placenta that are highly responsive to transferrin.     

Epidermal growth factor and transforming growth factor-alpha decrease gamma interferon receptors and induction of intercellular adhesion molecule (ICAM-1) on cultured keratinocytes.

The link between the epidermal keratinocytes of the skin and the activated T lymphocytes of the immune system is mediated by a variety of cytokines, including gamma interferon (IFN-gamma). We studied the influence of keratinocyte mitogens such as transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and somatomedin-C (SM-C) on the ligand binding of 32P-labeled IFN-gamma to cultured keratinocytes derived from normal appearing adult human skin. Keratinocytes placed in a medium devoid of mitogens become growth arrested, and these quiescent cells expressed 2.4 times (28,900 versus 12,200 sites/cell) as many high affinity IFN-gamma receptors (Kd = 0.22 nM) compared to keratinocytes which were actively growing in medium containing TGF-alpha (25 ng/ml) or EGF (10 ng/ml). The reduction in IFN-gamma receptor sites by TGF-alpha/EGF was mitogen specific, as adding SM-C (500 ng/ml) did not have any effect on ligand binding, although it similarly stimulated keratinocyte growth. The reduction in IFN-gamma receptors was time dependent, occurring primarily after 24-48 hours of change in tissue culture conditions. The reduction in the number of high affinity IFN-gamma receptors by TGF-alpha/EGF had immunobiological consequences, because quiescent keratinocytes in basal medium had an increased expression of HLA-DR and intercellular adhesion molecule-1 (ICAM-1) induced by IFN-gamma, compared to actively growing TGF-alpha/EGF treated keratinocytes. These results suggest that rapidly proliferating keratinocytes exposed to TGF-alpha/EGF but not SM-C are capable of altering their response to IFN-gamma by decreasing their number of cell surface high affinity receptors for IFN-gamma.     

Characterization and partial purification of keratinocyte growth factor from the hypothalamus

An extract of bovine hypothalamus is known to be mitogenic for human keratinocytes in vitro. In order to identify the responsible substance(s), biochemical characterization and subsequent bioassay of the extract in a serum-free culture system were performed. The keratinocyte growth-promoting activity of the hypothalamic extract was unaffected by heating (100 degrees C, 10 min); acidification to pH 3.3; or by exposure to lipase, RNAase, or proteolytic enzymes; but was abolished by alkalinization to pH 11. An approximate molecular weight of 1,700 daltons was determined by elution on a calibrated Sephadex G-25 column, and an approximate pl of 3.5 was determined by isoelectric focusing. Optimal concentrations of the crude extract (150-300 micrograms/ml) increased keratinocyte growth approximately 50-fold compared to control cultures lacking the extract. Partial purification resulted in a preparation biologically active at 30 ng/ml protein equivalent and was consistent with the presence of a single mitogen which we have termed keratinocyte growth factor (KGF). Mitogenic activity for human melanocytes, dermal fibroblasts, and endothelial cells, present in the crude hypothalamic extract, was lacking in heat-treated preparations that contained KGF. Optimal concentrations of purified epidermal growth factor and ethanolamine, the only remotely similar substances previously reported to augment keratinocyte growth in vitro, could not substitute for KGF in the serum-free culture system. Keratinocyte growth-promoting activity comparable to that observed in bovine hypothalamic extracts was present in human hypothalamic extracts prepared in the same manner.     

Characterization of baboon pregnancy-specific beta 1-glycoprotein

Immunostaining of baboon placental tissues with anti-human pregnancy-specific beta 1-glycoprotein (SPI) antibodies demonstrated that an SP1-like molecule was present in the syncytiotrophoblasts. Staining was observed on the membrane and in the cytoplasm, but the nucleus was devoid of any staining. Western blot analysis further demonstrated the presence of five protein species in baboon placental extract, whereas four protein bands were detected in human placental extract. Culture medium of baboon placental villi also contained five SP1-like molecules with sizes slightly different from those present in the placental extract. Amniotic fluid and culture medium of decidua basalis and chorioamniotic tissue contained lesser quantities and fewer species of SP1-like molecules. However, an 87 kDa band was present in all samples examined. Northern blot analysis of baboon placenta with a human placental SP1 cDNA probe demonstrated the presence of a 1.65 Kb band, whereas two hybridizing bands (1.65 Kb and 2.25 Kb) were present in human placenta. Southern blot analysis of baboon genomic DNA further demonstrated the presence of multiple bands hybridizing with a human placental SP1 cDNA probe. These results showed the presence in baboons of multiple genes encoding mRNAs and proteins highly similar to human placental SP1.