Bruce E. Magun

Prolactin receptors on human lymphocytes and their modulation by cyclosporine

Prolactin receptors have been identified for the first time on human peripheral blood lymphocytes. These receptors are present on T- and B-cells as well as monocytes. The specific binding of [125I]prolactin to these cells can be selectively enhanced at certain concentrations and blocked by higher concentrations of cyclosporine , a known immunosuppressive agent which inhibits the mitogenesis of T-cells. Prolactin also induces ornithine decarboxylase, a key growth regulatory enzyme, in lymphocytes. Therefore, we suggest that the lymphocyte prolactin receptor may be involved in regulating lymphocyte function, and that one of the actions of cyclosporine is to block this rather ubiquitously occurring receptor.

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.

Effects of Hyperthermia on Binding, Internalization, and Degradation of Epidermal Growth Factor

125 I-epidermal growth factor was used as a molecular probe to study the effects of hyperthermia and local anesthetics on cultured Rat-1 cells. Heating cells at 45°C for times up to 1 hr caused a continuous decrease in EGF binding. Scatchard analysis showed that the decreased binding resulted from a decrease in the affinity of the EGF receptors rather than from a decrease in receptor number. Whereas hyperthermia as high as 45°C for 30 min did not inhibit the process of receptor-mediated endocytosis of EGF, the degradation of internalized EGF was strongly inhibited at temperatures from 43 to 46°C. Exposure to 42°C had no effect on degradation. Because EGF degradation has been shown to occur in lysosomes, we compared the effects of heat to those caused by the local anesthetics procaine and lidocaine, which have been shown to prevent EGF degradation. The latter compounds, as well as other lysosomotropic amines, were similar to hyperthermia in their ability to permit accumulation of internalized EGF by preven...

Further purification of epidermal growth factor by high-performance liquid chromatography

Abstract Epidermal growth factor (EGF) purified by the method of Savage and Cohen ( J. Biol. Chem. 247, 7601–7611 (1972) using DEAE-cellulose chromatography as the final purification step was further resolved into two major uv-absorbing components by reverse-phase high-performance liquid chromatography (HPLC). Both components, referred to as α-EGF and β-EGF, competed with 125 I-labeled EGF for the EGF receptor, induced premature eye opening in neonatal mice, and had an amino acid composition similar to that published by Savage et al. ( J. Biol. Chem. 247, 7612–7621 (1972). β-EGF migrated slightly faster than α-EGF during sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. α-EGF was fourfold more potent than β-EGF and was 10-fold more potent than DEAE-purified EGF in stimulating DNA synthesis in quiescent Rat-1 cells. HPLC purification of EGF can replace the DEAE-cellulose chromatography step currently used and produces a more potent and less heterogeneous EGF species.

Identification of an epidermal growth factor-related transforming growth factor from rat fetuses.

Abstract An epidermal growth factor (EGF)-like transforming growth factor was identified in acid/ethanol extracts of 19-day-old rat embryos. This factor had an apparent molecular weight of 55,000 and possessed EGF-competing activity, stimulated DNA synthesis in quiescent Rat-1 fibroblasts, and induced progressive growth of untransformed cells in soft agar. The factor was trypsin sensitive and required intact disulfide bonds for colony stimulating activity. It was determined to be distinct from adult mouse submaxillary gland EGF based on its elution from BioGel P-60 column and its increased colony stimulating activity/unit of EGF-competing activity.

Binding of epidermal growth factor in rat pancreatic acini

Specific, saturable EGF receptors were demonstrated in isolated rat pancreatic acini. Binding of EGF to these receptors was one-half maximal at 20 min and maximal at 120 min. Scatchard analyses revealed a single order of binding sites with a Kd of 4.90 nM. Following binding, EGF was rapidly internalized and converted to two acidic species. EGF did not alter either basal amylase release or the rate of [3H]phenylalanine incorporation into TCA-precipitable protein. The finding of high affinity EGF receptors in pancreatic acinar cells supports the hypothesis that EGF participates in the long-term regulation of pancreatic exocrine function.

Dissociation of 68,000 M r Heat Shock Protein Synthesis from Thermotolerance Expression in Rat Fibroblasts

Rat embryonic fibroblasts growing exponentially at either 35, 37, or 39 degrees C were exposed to 42 degrees C for times up to 6 hr. Cell survival was unaffected by this heat shock in cultures growing at 39 degrees C but survival was decreased in a temperature dependent manner in cells growing at 37 or 35 degrees C. Exposure to 42 degrees C of cells previously adapted to 35 or 37 degrees C resulted in the induction of heat shock proteins (hsps) with apparent molecular weights of 68,000 (hsp 68), 70,000 (hsp 70), and 89,000 (hsp 89); cells previously adapted to 39 degrees C expressed all hsps except hsp 68. Inasmuch as the synthesis of certain hsps may function to protect cells from thermal damage, these data indicate that hsp 68 may not be required for this adaptation-related thermotolerant survival response. Hsp 68 may only be expressed in cells destined to die.

Binding and processing of epidermal growth factor in Panc-I human pancreatic carcinoma cells.

The binding of 125I-labeled epidermal growth factor (EGF) was studied in Panc-I human pancreatic carcinoma cells. At 37 degrees C, binding was rapid and associated with marked endocytosis of the ligand. Bound EGF was sequentially converted to a number of more acidic species as follows: pI 4.55 to pI 4.2, to pI 4.35, to pI 4.0. EGF internalization and processing were blocked at 4 degrees C. EGF did not alter cell growth when Panc-I cells were incubated in the presence of 2 to 10% serum. In contrast, when the serum concentration was lowered to 0.1%, EGF significantly enhanced cell replication after 6 days of culture.

Epidermal growth factor binding is altered in pancreatic acini from diabetic rats

The effects of streptozotocin-induced diabetes on 125I-labeled epidermal growth factor (EGF) binding were studied in rat pancreatic acini. 125I-EGF binding was one-half maximal at 20 min, and maximal at 90 min. Saturation data revealed a decreased binding capacity in diabetic acini when compared with normal acini. Insulin, in vivo, normalized the decreased binding capacity. 125I-EGF internalization was also decreased in diabetic rat acini. Further, the inhibitory effect of cholecystokinin-octapeptide (CCK8) on cell-associated 125I-EGF radioactivity was significantly greater in diabetic than in normal rat acini. These findings suggest that insulin deficiency may lead to defective regulation of the exocrine pancreas by EGF.

Internalization and processing of epidermal growth factor in aging human fibroblasts in culture

Tissue culture lines established from newborn human skin were used as a model system to study the effects of the mitogenic hormone epidermal growth factor (EGF) on the aging process. These cells demonstrated a finite life span in culture which is presumed to be related to the in vivo aging process. Fibroblasts aged in vitro demonstrated a reduction in their ability to respond to the mitogenic effects of EGF as compared to these cells at an earlier population doubling level (PDL). This decreased responsiveness was not due to a decrease in the number of EGF receptors/cells, as cells at late PDL possessed either the same or more EGF receptors than cells at an earlier PDL. In Rat-1 fibroblasts, 125I-labeled EGF is internalized following binding to the surface receptor, and is transported through intracellular organelles where it undergoes a series of modifications which result in acidification of the EGF (Matrisian et al., 1984, J. Biol. Chem. 259, 3047). It is not known whether this acidic processing of EGF is necessary for mitogenic activity. EGF internalization and processing were therefore examined in aging human fibroblasts to determine if the decrease in EGF responsiveness is due to an alteration in EGF processing. Human fibroblasts internalized and processed pI 4.55 125I-labeled EGF to the more acidic pI 4.2, 4.35, and 4.0 species in a manner similar to Rat-1 fibroblasts. The nature of the processed product and the time course of processing was the same in cells at early and late passages. We therefore conclude that the decreased responsiveness of aged cells to EGF is not due to a defect in the EGF-processing mechanism.