Prolactin increases the susceptibility of primary leukemia cells to NK and LAK effectors.

Our previous studies have shown that prolactin (PRL), a pituitary and lymphocyte hormone and a ligand of the cytokine/hemopoietin receptors (R) superfamily, acts synergistically with interleukin (IL)-2 on the development of lymphokine activated killer (LAK) cells and enhances the effects of GM-CSF and IL-3 on myeloid progenitors' proliferation and differentiation. More recently, we have demonstrated that GM-CSF and IL-3 increase the sensitivity of acute myeloid leukemic (AML) cells to LAK activity. Together, these findings have prompted us to study the role of PRL on the target arm of the LAK response. We show here that CD33+ blasts from AML patients express membrane PRL-R and that the PRL/PRL-R interaction is followed by increased susceptibility to natural killer (NK) (p < 0.02) and LAK (p < 0.001) cells. As predicted from the dimerization model of PRL-R and in agreement with previous reports, the response of AML blasts to PRL was bell-shaped with a trend peak at 25 ng/ml. Although enhanced lysis occurred at the target recognition level, it was not accompanied by changes in the MHC class I, cellular adhesion molecules, or myeloid differentiation antigens. Cell cycle recruitment and lysis increased concurrently in three cases studied, suggesting a modulatory action of PRL on the expression of putative cycle-related NK/LAK-target structures. Together, these data strengthen the role of PRL in the LAK response.

Biosynthesis of the precursor of a soluble human insulin receptor ectodomain in insect Sf9 cells infected with a recombinant baculovirus.

In contrast to transfected mammalian cells, insect Sf9 cells infected with a recombinant Baculovirus inefficiently process and secrete a soluble derivative of the extracellular domain of the human insulin receptor. The high-mannose form of the receptor precursor that accumulates intracellularly is not grossly aberrant or malfolded, as its interaction with a diverse panel of monoclonal antibodies are comparable to secreted precursor and proteolytically processed receptor, both of which bear partially trimmed oligosaccharide chains. Thus the inefficient step in the biosynthesis of this protein in Sf9 cells is either at, or just preceding, the trimming of its high-mannose oligosaccharide chains.

Secretion of the extracellular domain of the human insulin receptor from insect cells by use of a baculovirus vector.

To explore the utility of the baculovirus/insect-cell system for the expression of a soluble secreted human insulin-receptor (hIR) extracellular ligand-binding domain, we have engineered a recombinant virus encoding an hIR deletion mutant which is truncated eight residues from the beginning of the predicted transmembrane domain (i.e. 921 residues). Within 24 h after infection of Sf9 cells with virus, insulin-binding activity begins to accumulate in the culture medium, and reaches a maximum between 48 and 72 h. The intracellular transit and processing of this secreted receptor, designated 'AchIR01', is quite slow. After 24 h in pulse-chase experiments approximately 50% of the metabolically labelled protein is still inside the cell. This protein accumulates as a non-cleaved hIR precursor which is glycosylated, but the carbohydrate is entirely endoglycosidase H (endoH)-sensitive (i.e. high mannose). Approximately one-half of the receptor in the culture medium (i.e. approximately 25% of the total) is in the form of non-cleaved precursor, and about one half of its carbohydrate chains are now endoH-resistant. The remainder of the protein is proteolytically processed hIR (alpha-plus truncated beta-subunits). None of these hIR species exhibit O-linked carbohydrate. Only the processed form of the receptor in the medium binds insulin. This insulin-binding protein is secreted as a dimer (alpha beta)2, and binds insulin with an affinity which is comparable with that of both the wild-type hIR as well as the secreted form of the hIR expressed in mammalian cells. Despite the rather inefficient processing and altered glycosylation of the AchIR01 protein in insect cells, this high-affinity insulin-binding protein accumulates in the medium at levels (mg/litre) of about 100 times that achieved in a mammalian-cell system.

Anti-growth action on mouse mammary and prostate glands of a monoclonal antibody to prolactin receptor.

Monoclonal antibody (PrR-7A) against purified PRL receptor was used in the following studies. When PRL receptor was chromatographed on affinity columns containing PrR-7A antibody or monoclonal antibody against hemocyanin, which served as a control, PRL receptor was bound to the column containing PrR-7A antibody, but not to the column containing control antibody. When solubilized PRL receptor was incubated with PrR-7A antibody, the specific binding of the receptor was reduced 52%. Female mice were treated with the carcinogen, 7,12-dimethylbenz[a]anthracene, and during the succeeding 48 weeks were treated weekly with PrR-7A antibody or control antibody. In the control group 13% developed mammary carcinomas, and 16% developed moderate-to-severe intraductal hyperplasia. No mammary carcinomas were found in the mice treated with PrR-7A antibody, and only 8% of the mice had moderate-to-severe intraductal hyperplasia. Male mice made hyperprolactinemic by implanted pituitary glands were treated weekly with PrR-7A or control antibody. After 7 weeks of treatment, the mean weight of the prostates of mice treated with PrR-7A antibody was 8 +/- 1.1 mg (mean +/- SE), and that of mice treated with control antibody was 27 +/- 3.6 mg. Similar differences were seen in the protein and DNA content of the prostates. These results indicate that PrR-7A antibody is directed against PRL receptor and that immunization with this antibody reduces the incidence of PRL-dependent mammary tumors and preneoplastic ductal hyperplasia and prevents PRL-induced hyperplasia of the prostate.

Truncation of the ectodomain of the human insulin receptor results in secretion of a soluble insulin binding protein from transfected CHO cells.

The insulin receptor is an integral transmembrane glycoprotein comprised of two alpha-(approximately 135 kDa) and two beta-(approximately 95 kDa) subunits, which is synthesized as a single polypeptide chain precursor (alpha beta). The primary sequence of the human insulin receptor (hIR) protein, deduced from the nucleotide sequence of cloned human placental mRNAs, predicts two large domains (929 and 403 residues) on either side of a single membrane spanning domain (23 residues); each of these major domains has a distinct function (insulin binding and protein/tyrosine kinase activity, respectively). To experimentally test this deduced topology, and to explore the potential for independent domain function by the hIR extracellular domain, we have constructed an expression plasmid encoding an hIR deletion mutant which is truncated 8 residues from the beginning of the predicted transmembrane domain (i.e., 921 residues). This domain of the hIR is in fact processed into alpha- and truncated beta-subunits and secreted with high efficiency from transfected CHO cell lines which express this mutant hIR, and the protein accumulates as an (alpha beta)2 dimer in the medium. This molecule is recognized by a battery of 13 monoclonal antibodies to epitopes on the IR extracellular domain, four of which block insulin binding and two of which require the native conformation of the IR for recognition. Further, this domain binds insulin with an apparent dissociation constant comparable to that of the wild-type hIR. However, the secreted dimer displays a linear Scatchard plot, while that of the wild-type membrane-associated hIR is curvilinear.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased binding of epidermal growth factor in placentas from streptozotocin-diabetic rats.

Placentas from streptozotocin-diabetic rats have previously been shown to be morphologically and biochemically immature when compared with those of control rats. The binding of epidermal growth factor (EGF) to plasma membranes prepared from placentas of control and streptozotocin-diabetic fetuses has been characterized on days 17 and 21 of gestation. Results from competitive binding data analyzed by Scatchard analysis indicate the presence of a single class of receptors on day 17 (KD = 5.4 X 10(-10)) and the appearance of a second class of binding sites for 125I-EGF by day 21 (Kd = 3.5 X 10(-9)) in membranes from control fetuses. Placental membranes from diabetic fetuses show decreased specific binding (approximately 30%) on both days and the absence of a second class of binding sites on day 21 of gestation. Results from a radioreceptor assay indicate that the quantity of EGF in the serum of fetuses removed from control rats on day 21 is twofold greater than the quantity in serum of fetuses from diabetic rats. These data reveal a developmental increase in EGF-binding sites in the placenta of normal, near-term fetal rats, largely because of the appearance of a second class of binding sites with a lower affinity for EGF. The failure (or delay) of this second class to develop in the diabetic may be important for the control of maturation and growth of this tissue.

Influence of epidermal growth factor on fetal rat lung development in vitro.

Epidermal growth factor (EGF) has been shown to enhance cell multiplication or differentiation in a number of developing tissues. We have examined the effects of this growth factor on the biochemical development of explants of fetal rat lung, cultured in serum-free medium for 48 h. EGF enhanced the rate of choline incorporation into phosphatidylcholine and disaturated phosphatidylcholine in a dose dependent fashion. Half maximal stimulation occurred at a concentration of 1.0 nM, similar to the Kd for EGF binding to rat lung cell membranes. There was also significant stimulation of acetate incorporation into all phospholipids, particularly phosphatidylglycerol (539%), and increased distribution of radioactivity from acetate in this phospholipid fraction. Exposure to EGF stimulated PC synthesis in 18- and 19-day explants (term is 22 days) whereas maximal enhancement of DNA synthesis occurred after this time. This sequence differs from that observed during early embryonic development when EGF initially enhances cell multiplication. An additive interaction with regard to enhancement of PC synthesis was observed with EGF and thyroid hormone, but not EGF and dexamethasone. EGF had no effect on the activity of the enzymes of the choline incorporation pathway of phosphatidylcholine synthesis or on the activity of enzymes involved with acidic phospholipid synthesis. Fetal lung EGF content and EGF binding capacity were not increased by glucocorticoid treatment and similarly glucocorticoid binding capacity was not increased by EGF. These data indicate that EGF enhances fetal rat lung phospholipid synthesis in a dose-dependent manner and suggest that this is a direct effect on the lung tissue mediated by specific receptors.